Methods and agents for the diagnosis and treatment of hepatocellular carcinoma

ABSTRACT

The present invention relates to methods of diagnosing, and methods of treating, hepatocellular carcinoma in a subject. The invention also relates to polypeptide antagonists of PLVAP proteins, including humanized and chimeric antibodies that specifically bind PLVAP proteins, as well as compositions and kits comprising such polypeptide antagonists.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/126,734, filed Jul. 13, 2011, now U.S. Pat. No. 8,821,880, which isthe U.S. National Stage of International Application No.PCT/US2009/056382, filed Sep. 9, 2009, which designates the U.S., ispublished in English, and claims the benefit of U.S. ProvisionalApplication No. 61/197,650, filed Oct. 29, 2008.

The entire teachings of the above applications are incorporated hereinby reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

File name: 42611001022SubstSeqList.txt; created Sep. 25, 2014; 102 KB insize.

BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC) is the most frequent primary malignancyof the liver and is the fifth most common cancer in humans worldwide.HCC also is the fourth leading cause of cancer-related death (Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden:Globocan 2000. Int J Cancer 2001; 94: 153-156). In 1990, the WorldHealth Organization estimated that there were about 430,000 new cases ofliver cancer worldwide, and that a similar number of patients died thatyear as a result of this disease.

The pathogenesis of HCC has been associated with chronic hepatitis Bvirus (HBV) and hepatitis C virus (HCV) infections, as well ascirrhosis-inducing conditions of liver (Bruix J, et al. J Hepatol35:421-430, 2001; Bruix J, et al. Cancer Cell 5:215-219, 2004).Accordingly, the incidence of HCC is highest in East Asian countries,such as China, Hong Kong, Taiwan, Korea, and Japan, where HBV and HCVinfections are most prevalent (Bruix J, et al. Cancer Cell 5:215-219,2004; Haskell C M. Chapter 46 Liver: Natural History, Diagnosis andStaging in “Cancer Treatment” 5^(th) edition, W. B, Saunders Company,Philadelphia, editors: Haskell C M & Berek J S). However, the incidenceof HCC in western countries is steadily increasing (Parkin D M, et al.Int J Cancer 94; 153-156, 2001). Over the past decade, in the UnitedStates, HCC displayed the second highest increase in incidence, and thehighest increase in death rate, of all cancers (Ann Int Med 139:817-823,2003). Thus, in the United States and throughout the world, HCC is amajor cause of mortality and morbidity, and a significant economicburden due to hospital costs and loss of work by people with HCC.

Successful control of HCC requires correct diagnosis of the disease atan early stage of disease progression. However, distinguishing small HCCtumors from other malignant or non-malignant liver diseases, includingmetastatic tumors, cholangiocarcinoma, focal nodular hyperplasia,dysplastic and regenerating liver nodules, using current techniques,such as imaging studies, needle core biopsy and/or fine needleaspiration, has proven to be challenging (Ferrell L D, et al. Am J SurgPathol 17:1113-1123, 1993; Horigome H, et al. Hepato-Gatroenterology47:1659-1662, 2000; Kalar S, et al. Arch Pathol Lab Med 131:1648-1654,2007; Seki S, et al. Clin Cancer Res 6:3460-3473, 2000). Moreover,attempts to treat HCC therapeutically have been largely unsuccessful(Bruix J, et al. J Hepatol 35:421-430, 2001; Bruix J, et al. Cancer Cell5:215-219, 2004; Haskell C M. Chapter 46 Liver: Natural History,Diagnosis and Staging in “Cancer Treatment” 5^(th) edition, W. B,Saunders Company, Philadelphia, editors: Haskell C M & Berek J S;Szklaruk J, et al. AJR 180:441-453, 2003). As a result, despite activetherapy, the 5-year survival rate of patients with HCC in the U.S. isonly 10.5%, which is second in magnitude only to pancreatic cancer (ACSCancer Facts & Figures (2007)). Thus, there is an urgent need toidentify a more reliable marker to differentiate HCC from other liverpathologies and facilitate early detection of this disease. In addition,there is an urgent need to develop new and more-effective therapeuticagents for the treatment of HCC.

SUMMARY OF THE INVENTION

The present invention, in one embodiment, relates to a humanizedantibody that specifically binds human Plasmalemma Vesicle-AssociatedProtein (PLVAP), wherein the antibody comprises at least one heavy chainamino acid sequence selected from the group consisting of SEQ ID NO:66,SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100,SEQ ID NO:102 and a combination thereof; and at least one kappa lightchain amino acid sequence selected from the group consisting of SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, SEQ IDNO:108 and a combination thereof.

In another embodiment, the invention relates to a humanized antibodythat specifically binds human PLVAP, wherein the antibody comprises atleast one heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80,SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86 and a combination thereof; andat least one kappa light chain amino acid sequence selected from thegroup consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92 and a combination thereof.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising at least one humanized antibody that specificallybinds a PLVAP protein (e.g., a human PLVAP protein). In anotherembodiment, the pharmaceutical composition further comprises a secondtherapeutic agent, such as a chemotherapeutic agent.

In a further embodiment, the invention relates to an isolatedpolypeptide that specifically binds human PLVAP, comprising at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, SEQ ID NO:102 and a combination thereof and at least onekappa light chain amino acid sequence selected from the group consistingof SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108 and a combination thereof. In another embodiment,the polypeptide is a chimeric antibody.

In an additional embodiment, the invention provides an isolatedpolypeptide that specifically binds human PLVAP, comprising at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86 and a combination thereof and at least onekappa light chain amino acid sequence selected from the group consistingof SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92and a combination thereof. In another embodiment, the polypeptide is achimeric antibody.

In other embodiments, the invention relates to murine hybridoma KFCC-GY4(ATCC Patent Deposit Designation PTA-9963), cells thereof, andantibodies produced by murine hybridoma KFCC-GY4.

In yet other embodiments, the invention relates to murine hybridomaKFCC-GY5 (ATCC Patent Deposit Designation PTA-9964), cells thereof, andantibodies produced by murine hybridoma KFCC-GY5.

In yet another embodiment, the invention relates to a method of treatinghepatocellular carcinoma (HCC) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ahumanized antibody that specifically binds PLVAP. In a particularembodiment, the antibody is administered to the subject byintra-arterial infusion (e.g., hepatic arterial infusion, transarterialchemoembolization) and can inhibit tumor formation, tumor growth, tumorvascularization or tumor progression in the liver of the subject. Inanother embodiment, the PLVAP antagonist is administered in combinationwith a second therapeutic agent, such as a chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIG. 1 is a flow chart diagram depicting an algorithm for theidentification of genes that show extreme differential expressionbetween tumor and adjacent non-tumorous tissues.

FIG. 2 is a graph depicting PLVAP gene expression intensities in pairedHCC (PHCC) and adjacent non-tumorous liver tissue (PN) samples (n=18),as well as unpaired HCC samples (n=82) as determined by mRNA transcriptprofiling using Affymetrix gene chips.

FIG. 3A is a graph depicting relative PLVAP expression quantities inpaired HCC (PHCC) and adjacent non-tumorous liver tissue (PN) samples asdetermined by Taqman quantitative RT-PCR. PLVAP mRNA levels aresignificantly higher in HCC relative to non-tumorous liver tissues.

FIG. 3B is a graph depicting PLVAP gene expression intensities in 18paired HCC (PHCC) and adjacent non-tumorous liver tissue (PN) samples asdetermined by microarray analysis. PLVAP transcript levels were higherin HCC than in adjacent non-tumorous liver tissue from each individualfor all individuals tested except one.

FIGS. 4A and 4B show the nucleotide sequence (SEQ ID NO:1) and thededuced amino acid sequence (SEQ ID NO:2) of the His-tagged humanPLVAP₅₁₋₄₄₂ protein recombinant fusion protein used to generate mouseanti-PLVAP polyclonal antisera.

FIG. 5 is an image of a Western blot depicting the detection ofrecombinant PLVAP protein before and after thrombin digestion to removethe His tag. Arrows to the left of the blot indicate the locations ofHis-PLVAP and PLVAP on the blot. The numbers to the left of the blotindicate the positions of molecular weight standards.

FIG. 6A is a graph depicting the presence of significant relativequantities of PLVAP mRNA in HCC endothelial cells obtained bylaser-capturing microdissection from two HCC tissue samples (Sample A(black) and Sample B (gray)) as determined by two-step real-timequantitative RT-PCR. Dashed lines represent Taqman quantitative RT-PCRsignals from beta-actin mRNA in the same samples used for quantitativeRT-PCR of PLVAP mRNA. The results indicate presence of readilymeasurable PLVAP mRNA in the dissected endothelial cells (solid lines).

FIG. 6B is a graph depicting the absence of significant relativequantities of PLVAP mRNA in cells obtained by laser-capturingmicrodissection from non-tumorous liver tissue adjacent to HCC tissue intwo HCC samples (Sample A (black) and Sample B (gray)) as determined bytwo-step Taqman real-time quantitative RT-PCR. The results indicate nodetectable (solid black line) and barely detectable (solid gray line)PLVAP mRNA in the dissected cells.

FIG. 6C is a graph depicting the relative quantities of PLVAP mRNA inHCC tumor cells obtained by laser-capturing microdissection from two HCCtissue samples (Sample A (black) and Sample B (gray)) as determined bytwo-step Taqman real-time quantitative RT-PCR. The results indicatepresence of very small amounts of PLVAP mRNA (solid lines) in thedissected HCC cells due to unavoidable minor contamination from portionof vascular endothelial cells attached to the dissected HCC cells.

FIG. 7 is a graph depicting anti-PLVAP antibody titer in mouse antiserumraised against recombinant PLVAP₅₁₋₄₄₂ protein as determined by ELISA.

FIGS. 8A-8F are images showing sections of formalin-fixed paired HCC(FIGS. 8A, 8C, and 8E) and adjacent non-tumorous liver tissues (FIGS.8B, 8D, and 8F) from three patients with hepatocellular carcinoma thatwere stained immunohistochemically using anti-PLVAP polyclonal antiserato detect localization of PLVAP protein. Paired tissues are shown inFIGS. 8A and 8B; FIGS. 8C and 8D; and FIGS. 8E and 8F. PLVAP protein,which appears as a brown stain (arrows) in the HCC images, was detectedonly in capillary endothelial cells of hepatocellular carcinomas (FIGS.8A, 8C, and 8E). No detectable PLVAP was present in non-tumorous livertissue (FIGS. 8B, 8D, and 8F).

FIGS. 9A-9F are images showing sections of formalin-fixed HCC (FIGS. 9A,9C, 9E and 9F) and non-tumorous liver tissues (FIGS. 9B and 9D) fromthree additional patients with hepatocellular carcinoma that werestained immunohistochemically using anti-PLVAP polyclonal antisera todetect localization of PLVAP protein. FIGS. 9A and 9B and FIGS. 9C and9D show paired tissue samples of HCC and adjacent non-tumorous livertissue. PLVAP protein, which appears as a brown stain (arrows) in theHCC images, was detected only in capillary endothelial cells ofhepatocellular carcinomas (FIGS. 9A, 9C, 9E and 9F). No detectable PLVAPwas present in non-tumorous liver tissue (FIGS. 9B and 9D).

FIGS. 10A-10F are images showing sections of formalin-fixed focalnodular hyperplasia tissues from six different patients that werestained immunohistochemically using anti-PLVAP polyclonal antisera todetect localization of PLVAP protein. PLVAP protein was not detected inendothelial cells lining the vascular sinusoids/capillary ofnon-tumorous liver tissues of focal nodular hyperplasia. Some positivestaining (dark gray) was noted in epithelial cells of bile ducts (FIGS.10A, 10D and 10F) and vessels of portal tracts (FIGS. 10D and 10F), butnot in the endothelial cells of liver parenchyma. The positive stainingof bile duct epithelial cells was due to binding of non-specificantibodies in the PLVAP antiserum.

FIGS. 11A and 11B are images showing sections of formalin-fixed tissuefrom two patients with hepatic hemangioma that were stainedimmunohistochemically with anti-PLVAP polyclonal antiserum. Endotheliallining cells of hepatic hemangioma did not show significant expressionof PLVAP protein.

FIGS. 12A and 12B are images showing sections of formalin-fixed tissuefrom two patients with chronic active hepatitis B that were stainedimmunohistochemically with anti-PLVAP polyclonal antiserum. PLVAPprotein was not detected in endothelial cells lining the vascularsinusoids/capillary of non-tumorous liver tissues from chronic hepatitisB patients.

FIGS. 13A-13D are images showing sections of formalin-fixed tissue fromthree different patients with chronic active hepatitis C that werestained immunohistochemically with anti-PLVAP polyclonal antiserum. Thetissue sections shown in FIGS. 13B and 13D are from the same patient.PLVAP protein was not detected in endothelial cells lining the vascularsinusoids/capillary of non-tumorous liver tissues from chronic hepatitisC patients.

FIGS. 14A-14D are images showing sections of formalin-fixed tissue fromthree different patients with metastatic liver cancers that were stainedimmunohistochemically with anti-PLVAP polyclonal antiserum. The tissuesections are from patients with metastatic colorectal adenocarcinoma(FIG. 14A), intrahepatic cholangiocarcinoma (FIGS. 14B and 14C) ormetastatic ovarian carcinoma (FIG. 14D). The tissue sections shown inFIGS. 14B and 14C are from the same patient. PLVAP protein was notdetected in endothelial cells lining the vascular sinusoids/capillary ofmetastatic cancer tissues.

FIG. 15A shows the nucleotide gene (top) (SEQ ID NO:3) and deduced aminoacid (middle) (SEQ ID NO:4) sequences of the V_(H) domain of monoclonalantibody KFCC-GY4. The sequence of amino acid residues in CDRs 1 (SEQ IDNO:5), 2 (SEQ ID NO:6) and 3 (SEQ ID NO:7) also are indicated (bottom).

FIG. 15B shows the nucleotide gene (top) (SEQ ID NO:8) and deduced aminoacid (middle) (SEQ ID NO:9) sequences of the V_(L) domain of monoclonalantibody KFCC-GY4. The sequence of amino acid residues in CDRs 1 (SEQ IDNO:10), 2 (SEQ ID NO:11) and 3 (SEQ ID NO:12) also are indicated(bottom).

FIG. 16A shows the nucleotide gene (top) (SEQ ID NO:13) and deducedamino acid (middle) (SEQ ID NO:14) sequences of the V_(H) domain ofmonoclonal antibody KFCC-GY5. The sequence of amino acid residues inCDRs 1 (SEQ ID NO:15), 2 (SEQ ID NO:16) and 3 (SEQ ID NO:17) also areindicated (bottom).

FIG. 16B shows the nucleotide gene (top) (SEQ ID NO:18) and deducedamino acid (middle) (SEQ ID NO:19) sequences of the V_(L) domain ofmonoclonal antibody KFCC-GY5. The sequence of amino acid residues inCDRs 1 (SEQ ID NO:20), 2 (SEQ ID NO:21) and 3 (SEQ ID NO:22) also areindicated (bottom).

FIG. 17 is a graph depicting the binding of KFCC-GY4 (open circles) andKFCC-GY5 (filled circles) monoclonal antibodies to recombinant PLVAPprotein at various antibody concentrations, as determined by ELISA.

FIG. 18 is an immunoblot showing that KFCC-GY4 and KFCC-GY5 monoclonalantibodies can detect 5 ng of recombinant PLVAP protein. Lane 1:molecular weight standard; Lane 2: immunoblot with KFCC-GY4 monoclonalantibody; Lane 3: immunoblot with KFCC-GY5 monoclonal antibody. Themolecular weight of recombinant PLVAP protein is 45 kD.

FIGS. 19A and 19C are Coomassie blue-stained SDS acrylamide gels. Lane1: molecular weight standard; Lane 2: hydrophobic membrane proteinsextracted with TX-114 from human umbilical cord vascular endothelialcells that had been stimulated with VEGF (40 ng/ml) for 72 hours beforeextraction.

FIG. 19B is an immunoblot wherein the extract shown in Lane 2 of FIG.19A was probed with KFCC-GY4 monoclonal antibodies. Lane 1: molecularweight standard; Lane 2: hydrophobic membrane proteins extracted withTX-114 from human umbilical cord vascular endothelial cells that hadbeen stimulated with VEGF (40 ng/ml) for 72 hours before extraction.

FIG. 19D is an immunoblot wherein the extract shown in Lane 2 of FIG.19C was probed with KFCC-GY-5 monoclonal antibodies. Lane 1: molecularweight standard; Lane 2: hydrophobic membrane proteins extracted withTX-114 from human umbilical cord vascular endothelial cells that hadbeen stimulated with VEGF (40 ng/ml) for 72 hours before extraction.

FIG. 20A is a fluorescence micrograph depicting immunofluorescencestaining of human vascular endothelial cells (HUVEC) with control normalmouse IgG. Nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI). Magnification=600×.

FIG. 20B is a fluorescence micrograph depicting immunofluorescencestaining of human vascular endothelial cells (HUVEC) with monoclonalantibody to von Willebrand factor (VWF). VWF is a positive marker forhuman vascular endothelial cells. Nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI). Magnification=600×.

FIG. 20C is a fluorescence micrograph depicting immunofluorescencestaining of human vascular endothelial cells (HUVEC) with KFCC-GY4monoclonal antibody to PLVAP. KFCC-GY4 monoclonal anti-PLVAP antibodiesreacted positively with human vascular endothelial cells. Nuclei werestained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification=600×.

FIG. 20D is a fluorescence micrograph depicting immunofluorescencestaining of human vascular endothelial cells (HUVEC) with KFCC-GY5monoclonal antibody to PLVAP. KFCC-GY5 monoclonal anti-PLVAP antibodiesreacted positively with human vascular endothelial cells. Nuclei werestained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification=600×.

FIG. 21A is a light micrograph of a section of formalin-fixed hepatomatissue embedded in a paraffin block that was stained with KFCC-GY5monoclonal anti-PLVAP antibodies. A strong PLVAP signal (dark graystain) was detected in vascular endothelial cells of hepatoma.Magnification is 100×.

FIG. 21B is a light micrograph of a section of formalin-fixed hepatomatissue from the same patient as the sample shown in FIG. 21A that wasstained with KFCC-GY4 monoclonal anti-PLVAP antibodies. A moderate PLVAPsignal (light gray stain) was detected in vascular endothelial cells ofhepatoma. Magnification is 100×.

FIG. 21C is a light micrograph of a section of formalin-fixed hepatomatissue from a different patient than the samples shown in FIGS. 21A and21B that was stained with KFCC-GY5 monoclonal anti-PLVAP antibodies. Astrong PLVAP signal (dark gray stain) was detected in vascularendothelial cells. Magnification is 100×.

FIG. 21D is a light micrograph of a section of formalin-fixed hepatomatissue from the same patient as the sample shown in FIG. 21C embedded ina paraffin block that was stained with KFCC-GY4 monoclonal anti-PLVAPantibodies. A moderate PLVAP signal (light gray stain) was detected invascular endothelial cells, indicating that KFCC-GY4 monoclonalantibodies bind the PLVAP antigen less well than KFCC-GY5 antibodies.Magnification is 100×.

FIGS. 22A-22H are light micrographs of sections of hepatoma tissues(FIGS. 22A, 22C, 22E, and 22G) and adjacent non-tumorous liver tissues(FIGS. 22B, 22D, 22F, and 22H) from four different randomly selectedhepatoma patients. The sections were stained with KFCC-GY5 monoclonalanti-PLVAP antibodies. PLVAP signal (gray stain) was detected invascular endothelial cells of hepatoma tissue, but not in vascularendothelial cells non-tumorous liver tissue. Magnification is 100×.FIGS. 22A and 22B, 22C and 22D, 22E and 22F, and 22G and 22H representthe four sets of paired hepatoma and non-tumorous liver tissues.

FIG. 23A is a fluorescence micrograph depicting human vascularendothelial cells (HUVECs) that were stained with control mouse IgG.Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

FIG. 23B is a fluorescence micrograph depicting human vascularendothelial cells (HUVECs) that were stained with KFCC-GY4 monoclonalantibody to PLVAP. KFCC-GY4 monoclonal anti-PLVAP antibodies reactedpositively with the surfaces of the human vascular endothelial cells.Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

FIG. 23C is a fluorescence micrograph depicting human vascularendothelial cells (HUVECs) that were stained with KFCC-GY5 monoclonalantibody to PLVAP. KFCC-GY5 monoclonal anti-PLVAP antibodies reactedpositively with the surfaces of the human vascular endothelial cells.Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

FIG. 24 shows the amino acid sequence of human PLVAP protein (GENBANK®Accession No. NP_112600; SEQ ID NO:23).

FIGS. 25A and 25B show the nucleotide sequence of full-length humanPLVAP cDNA (GENBANK® Accession No. NM_031310; SEQ ID NO:24).

FIG. 26 is a table indicating PLVAP expression in vascular endothelialcells in various normal tissues and organs in humans and two non-humanprimates, as determined by immunohistochemistry using KFCC-GY4 and Gy5antibodies.

FIGS. 27A-27F2 show normal human and monkey tissues that wereimmunohistochemically stained with KFCC-GY4, KFCC-GY5 and anti-CD34monoclonal antibodies (mAbs). Arrows point to capillary endothelialcells that express PLVAP in the respective tissues.

FIG. 27A shows a section of human adrenal gland tissue that has beenstained with the KFCC-GY4 mAb. FIG. 27B shows a section of human adrenalgland tissue that has been stained with the anti-human CD34 mAb, whichrecognizes CD34, a marker for endothelial cells. FIG. 27C shows asection of adrenal gland tissue from cynomolgus monkey that has beenstained with the KFCC-GY4 mAb. FIG. 27D shows a section of adrenal glandtissue from rhesus monkey that has been stained with the KFCC-GY4 mAb.

FIG. 27E shows a section of human adrenal gland tissue that has beenstained with the KFCC-GY5 mAb. FIG. 27F shows a section of human adrenalgland tissue that has been stained with the anti-human CD34 mAb, whichrecognizes CD34, a marker for endothelial cells. FIG. 27G shows asection of adrenal gland tissue from cynomolgus monkey that has beenstained with the KFCC-GY5 mAb. FIG. 27H shows a section of adrenal glandtissue from rhesus monkey that has been stained with the KFCC-GY5 mAb.

FIG. 27I shows a section of human kidney tissue that has been stainedwith the KFCC-GY4 mAb. FIG. 27J shows a section of human kidney tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. FIG. 27K shows a section of kidneytissue from cynomolgus monkey that has been stained with the KFCC-GY4mAb. FIG. 27L shows a section of kidney tissue from rhesus monkey thathas been stained with the KFCC-GY4 mAb. KFCC-GY4 mAb stains capillaryendothelial cells between renal tubules and does not stain endothelialcells in glomeruli. In contrast, Anti-CD34 antibody stains positivelycapillary endothelial cells between renal tubules and in glomeruli.

FIG. 27M shows a section of human kidney tissue that has been stainedwith the KFCC-GY5 mAb. FIG. 27N shows a section of human kidney tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. FIG. 27O shows a section of kidneytissue from cynomolgus monkey that has been stained with the KFCC-GY5mAb. FIG. 27P shows a section of kidney tissue from rhesus monkey thathas been stained with the KFCC-GY5 mAb. Like KFCC-GY4 mAb, KFCC-GY5 mAbstains capillary endothelial cells between renal tubules and does notstain endothelial cells in glomeruli. In contrast, Anti-CD34 antibodystains positively capillary endothelial cells between renal tubules andin glomeruli.

FIG. 27Q shows a section of human brain tissue that has been stainedwith the KFCC-GY4 mAb. FIG. 27R shows a section of human brain tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. Vascular endothelial cells ofbrain stained positively for CD34 endothelial marker (arrows). FIG. 27Sshows a section of brain tissue from cynomolgus monkey that has beenstained with the KFCC-GY4 mAb. FIG. 27T shows a section of brain tissuefrom rhesus monkey that has been stained with the KFCC-GY4 mAb. Bothhuman and monkey brain endothelial cells do not express PLVAP.

FIG. 27U shows a section of human brain tissue that has been stainedwith the KFCC-GY5 mAb. FIG. 27V shows a section of human brain tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. Vascular endothelial cells ofbrain are stained positively for CD34 endothelial marker (arrows). FIG.27W shows a section of brain tissue from cynomolgus monkey that has beenstained with the KFCC-GY5 mAb. FIG. 27X shows a section of brain tissuefrom rhesus monkey that has been stained with the KFCC-GY5 mAb. Bothhuman and monkey brain endothelial cells do not express PLVAP.

FIG. 27Y shows a section of human liver tissue that has been stainedwith the KFCC-GY4 mAb. FIG. 27Z shows a section of human liver tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. Vascular endothelial cells ofbrain stained positively for CD34 endothelial marker (arrows). FIG. 27A2shows a section of liver tissue from cynomolgus monkey that has beenstained with the KFCC-GY4 mAb. FIG. 27B2 shows a section of liver tissuefrom rhesus monkey that has been stained with the KFCC-GY4 mAb. KFCC-GY4mAb does not react with endothelial cells of liver sinusoid (thinarrows) and central vein (thick arrows) in human, cynomolgus monkey andrhesus monkey.

FIG. 27C2 shows a section of human liver tissue that has been stainedwith the KFCC-GY5 mAb. FIG. 27D2 shows a section of human liver tissuethat has been stained with the anti-human CD34 mAb, which recognizesCD34, a marker for endothelial cells. Vascular endothelial cells ofbrain stained positively for CD34 endothelial marker (arrows). FIG. 27E2shows a section of liver tissue from cynomolgus monkey that has beenstained with the KFCC-GY4 mAb. FIG. 27F2 shows a section of liver tissuefrom rhesus monkey that has been stained with the KFCC-GY5 mAb. KFCC-GY5mAb does not react with endothelial cells of liver sinusoid (thinarrows) and central vein (thick arrows) in human, cynomolgus monkey andrhesus monkey.

FIG. 28A shows a Coomassie blue-stained SDS-PAGE gel of total proteinextract from 1×10⁸ E. coli expressing His-tagged PLVAP51-442 (Lane 1);His-tagged PLVAP282-442 (Lane 2); His-tagged PLVAP51-292 (Lane 3); orHis-tagged CEACAM6 (Lane 4). Molecular weight protein standards areresolved in Lane M.

FIG. 28B shows an immunoblot that was probed with mouse KFCC-GY4 mAb todetect PLVAP proteins in total protein extract from 1×10⁸ E. coliexpressing His-tagged PLVAP51-442 (Lane 1); His-tagged PLVAP282-442(Lane 2); His-tagged PLVAP51-292 (Lane 3); or His-tagged CEACAM6 (Lane4). Molecular weight protein standards are resolved in Lane M.

FIG. 28C shows an immunoblot that was probed with mouse KFCC-GY5 mAb todetect PLVAP proteins in total protein extract from 1×10⁸ E. coliexpressing His-tagged PLVAP51-442 (Lane 1); His-tagged PLVAP282-442(Lane 2); His-tagged PLVAP51-292 (Lane 3); or His-tagged CEACAM6 (Lane4). Molecular weight protein standards are resolved in Lane M.

FIG. 28D shows an immunoblot that was probed with anti-His tag antibodyto detect His-tagged PLVAP proteins in total protein extract from 1×10⁸E. coli expressing His-tagged PLVAP51-442 (Lane 1); His-taggedPLVAP282-442 (Lane 2); His-tagged PLVAP51-292 (Lane 3); or His-taggedCEACAM6 (Lane 4). Molecular weight protein standards are resolved inLane M.

FIG. 29 is a bar graph showing binding of KFCC-GY4 monoclonal antibody(mAb) to PLVAP that was captured first by KFCC-GY5 mAb. ELISA was usedfor the study. Each value is a mean of duplicates.

FIG. 30 is a bar graph depicting additive binding of fully humanizedcomposite monoclonal antibodies derived from KFCC-GY4 (CSR01-VH5NK2) andKFCC-GY5 (CAS02-VH5NK3) to PLVAP protein. The values are an average ofduplicates.

FIG. 31 shows a Coomassie blue-stained SDS-PAGE gel of protein-Apurified chimeric KFCC-GY4 and KFCC-GY5 antibodies after reduction ofdisulfide bonds. Lane 1: Precision plus protein standards (Bio-Rad);Lane 2: 1.0 μg chimeric KFCC-GY4 antibody; Lane 3: 1.0 μg chimericKFCC-GY5 antibody; Lane 4: Precision plus protein standards (Bio-Rad).

FIGS. 32A-1 to 32A-4, 32B-1 to 32B-3, 32C-1 to 32C-3, 32D-1 to 32D-5,32E-1 to 32E-3, 32F-1 to 32F-2 and 32G show the nucleotide sequence ofthe pANT12-based plasmid vector encoding the KFCC-GY4 VH Chimera and thededuced amino acid sequence of the chimera.

FIGS. 33A-1 to 33A-4, 33B-1 to 33B-3, 33C-1 to 33C-5 and 33D-1 to 33D-4show the nucleotide sequence of the pANT13-based plasmid vector encodingthe KFCC-GY4 VK Chimera and the deduced amino acid sequence of thechimera.

FIGS. 34A-1 to 34A-4, 34B-1 to 34B-3, 34C-1 to 34C-4, 34D-1 to 34D-5,34E-1 to 34E-4 and 34F-1 to 34F-3 show the nucleotide sequence of thepANT12-based plasmid vector encoding the KFCC-GY5 VH Chimera and thededuced amino acid sequence of the chimera.

FIGS. 35A-1 to 35A-4, 35B-1 to 35B-4, 35C-1 to 35C-3 and 35D-1 to 35D-4show the nucleotide sequence of the pANT13-based plasmid vector encodingthe KFCC-GY5 VK Chimera and the deduced amino acid sequence of thechimera.

FIG. 36 is a graph depicting the results of a KFCC-GY4 antibodycompetition ELISA in which a dilution series of chimeric and murineKFCC-GY4 antibodies were tested against a fixed concentration ofbiotinylated-GY4 for binding to PLVAP. Binding of biotinylated antibodydecreases with increasing amounts of chimeric and control murineantibodies.

FIG. 37 is a graph depicting the results of a KFCC-GY5 antibodycompetition ELISA in which a dilution series of chimeric and murineKFCC-GY5 antibodies were tested against a fixed concentration ofbiotinylated-GY5 for binding to PLVAP. Binding of biotinylated antibodydecreases with increasing amounts of chimeric and control murineantibodies.

FIGS. 38A-38E show the nucleotide and amino acid sequences of thevariable domains of heavy and light chains from the humanized antibodiesderived from chimeric KFCC-GY4 antibody CSR01. FIG. 38A shows thenucleotide and amino acid sequences of the kappa light chain CSR01-VK1(SEQ ID NOS:59 and 60). FIG. 38B shows the nucleotide and amino acidsequences of the kappa light chain CSR01-VK2 (SEQ ID NOS:61 and 62).FIG. 38C shows the nucleotide and amino acid sequences of the kappalight chain CSR01-VK3 (SEQ ID NOS:63 and 64). FIG. 38D shows thenucleotide and amino acid sequences of the heavy chain CSR01-VH4 (SEQ IDNOS:65 and 66). FIG. 38E shows the nucleotide and amino acid sequencesof the heavy chain CSR01-VH5 (SEQ ID NOS:67 and 68). The amino acidsequences of the CDRs are underlined. The amino acids that are alteredto reduce potential antigenicity are shown in a square box.

FIGS. 39A-39D show the nucleotide and amino acid sequences of thevariable domains of heavy and light chains from the humanized antibodiesderived from chimeric KFCC-GY5 antibody CSR02. FIG. 39A shows thenucleotide and amino acid sequences of the kappa light chain CSR02-VK2(SEQ ID NOS:69 and 70). FIG. 39B shows the nucleotide and amino acidsequences of the kappa light chain CSR02-VK3 (SEQ ID NOS:71 and 72).FIG. 39C shows the nucleotide and amino acid sequences of the heavychain CSR02-VH4 (SEQ ID NOS:73 and 74). FIG. 39E shows the nucleotideand amino acid sequences of the heavy chain CSR02-VH5 (SEQ ID NOS:75 and76). The amino acid sequences of the CDRs are underlined. The aminoacids that are altered to reduce potential antigenicity are shown in asquare box.

FIG. 40 is a flowchart diagram depicting the derivation of fullyhumanized anti-human PLVAP composite monoclonal antibodies (mAb) frommurine KFCC-GY4 and KFCC-GY5 mAbs.

FIG. 41A depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable heavy chain VH variant 1 (SEQ ID NOS:77 and 78). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 41B depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable heavy chain VH variant 2 (SEQ ID NOS:79 and 80). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 41C depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable heavy chain VH variant 3 (SEQ ID NOS:81 and 82). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 41D depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable heavy chain VH variant 4 (SEQ ID NOS:83 and 84). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 41E depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable heavy chain VH variant 5 (SEQ ID NOS:85 and 86). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 42A depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable light chain VK variant 1 (SEQ ID NOS:87 and 88). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 42B depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable light chain VK variant 2 (SEQ ID NOS:89 and 90). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 42C depicts the nucleotide and deduced amino acid sequences of theKFCC-GY5 variable light chain VK variant 3 (SEQ ID NOS:91 and 92). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 43A depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable heavy chain VH variant 1 (SEQ ID NOS:93 and 94). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 43B depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable heavy chain VH variant 2 (SEQ ID NOS:95 and 96). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 43C depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable heavy chain VH variant 3 (SEQ ID NOS:97 and 98). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 43D depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable heavy chain VH variant 4 (SEQ ID NOS:99 and 100). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 43E depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable heavy chain VH variant 5 (SEQ ID NOS:101 and 102). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 44A depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable light chain VK variant 1 (SEQ ID NOS:103 and 104). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 44B depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable light chain VK variant 2 (SEQ ID NOS:105 and 106). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 44C depicts the nucleotide and deduced amino acid sequences of theKFCC-GY4 variable light chain VK variant 3 (SEQ ID NOS:107 and 108). CDRnucleotide and protein sequences are lightly shaded. Variant amino acidschanged from the original hybridoma sequence are underlined.

FIG. 45 shows alignments of variable domain sequences of humanizedKFCC-GY4 antibody variant heavy chains (top alignment) and Kappa lightchains (bottom alignment).

FIG. 46 shows alignments of variable domain sequences of humanizedKFCC-GY5 antibody variant heavy chains (top alignment) and Kappa lightchains (bottom alignment).

FIG. 47A shows a Coomassie Blue-stained SDS-PAGE gel of purifiedhumanized KFCC-GY4 antibodies after reduction of disulfide bonds. Lane1: Precision Plus marker (Biorad); Lane 2: 1.0 μg VH4/VK2 IgG4; Lane 3:1.0 μg VH4/VK3 IgG4; Lane 4: 1.0 μg VH5/VK1 IgG4; Lane 5: 1.0 μg VH5/VK2IgG4; Lane 6: 1.0 μg VH5/VK3 IgG4; Lane 7: Precision Plus marker.

FIG. 47B shows a Coomassie Blue-stained SDS-PAGE gel of purifiedhumanized KFCC-GY5 antibodies after reduction of disulfide bonds. Lane1: Precision Plus marker; Lane 2: 1.0 μg VH4/VK2 IgG4; Lane 3: 1.0 μgVH4/VK3 IgG4; Lane 4: 1.0 μg VH5/VK2 IgG4; Lane 5: 1.0 μg VH5/VK3 IgG4;Lane 6: Precision Plus marker.

FIG. 48 is a graph depicting the results of a PLVAP competition ELISAillustrating the binding of purified variant KFCC-GY4 humanizedantibodies that were mixed with a fixed concentration of competitorbiotinylated-murine KFCC-GY4 antibody to PLVAP protein at varyingconcentrations.

FIG. 49 is a graph depicting the results of a PLVAP competition ELISAillustrating the binding of purified variant KFCC-GY5 humanizedantibodies that were mixed with a fixed concentration of competitorKFCC-GY5 antibody to PLVAP protein at varying concentrations.

FIG. 50 depicts a titration curve illustrating binding of both chimericand fully humanized composite anti-PLVAP monoclonal antibodies to PLVAP,as determined by ELISA.

FIGS. 51A-51C are images of human umbilical cord vascular endothelialcells that have been stained with murine KFCC-GY4 and KFCC-GY5monoclonal antibodies in immunofluorescence studies. FIG. 51A showsimmunofluorescence staining of human umbilical cord vascular endothelialcells with murine KFCC-GY4 mAbs. FIG. 51B shows immunofluorescencestaining of human umbilical cord vascular endothelial cells with murineKFCC-GY5 mAbs. FIG. 51C shows immunofluorescence staining of humanumbilical cord vascular endothelial cells with mouse IgG as a negativecontrol.

FIGS. 52A-52G are images of human umbilical cord vascular endothelialcells that have been stained with chimeric or humanized KFCC-GY4 (CSR01)antibodies in immunofluorescence studies. FIG. 52A: chimeric KFCC-GY4mAb; FIG. 52B: CSR01-VH4NK2; FIG. 52C: CSR01-VH4NK3; FIG. 52D:CSR01-VH5NK1; FIG. 52E: CSR01-VH5/VK2; FIG. 52F: CSR01-VH5/VK3; FIG.52G: human IgG.

FIGS. 53A-53F are images of human umbilical cord vascular endothelialcells that have been stained with chimeric or humanized KFCC-GY5antibodies in immunofluorescence studies. FIG. 53A: chimeric KFCC-GY5(CSR02) mAb; FIG. 53B: CSR02-VH4NK2; FIG. 53C: CSR02-VH4/VK3; FIG. 53D:CSR02-VH5NK2; FIG. 53E: CSR02-VH5NK3; FIG. 53F: human IgG.

FIG. 54 is a graph depicting the detection of PLVAP protein in two HCCpatient serum samples and in serially diluted PLVAP standards (1000ng/ml to 10 ng/ml). No PLVAP was detected in two normal serum samples.Serum samples were obtained from two patients with hepatocellularcarcinoma (HCC-63 and HCC-82) and two normal adults (Normal-13 andNormal-14). Serum samples were assayed at 2-fold and 4-fold dilutions.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

DEFINITIONS

As used herein, the terms “Plasmalemma Vesicle-Associated Protein,”“PLVAP,” and “PV-1” refer to a naturally occurring or endogenous PLVAP(e.g., mammalian, human) protein, and to proteins having an amino acidsequence that is the same or substantially the same as that of naturallyoccurring or endogenous PLVAP protein (e.g., recombinant proteins,synthetic proteins). Accordingly, the terms “PlasmalemmaVesicle-Associated Protein,” “PLVAP,” and “PV-1,” which are usedinterchangeably herein, include polymorphic or allelic variants andother isoforms of a PLVAP protein produced by, e.g., alternativesplicing or other cellular processes, that occur naturally in mammals(e.g., humans). Preferably, the PLVAP protein is a human protein thathas the amino acid sequence of SEQ ID NO:23 (see GENBANK® Accession No.NP_112600 and FIG. 24).

As defined herein, a “PLVAP antagonist” is an agent (e.g., antibody,small molecule, peptide, peptidomimetic, nucleic acid) that, in oneembodiment, inhibits (e.g., reduces, prevents) an activity of a PLVAPprotein; or, in another embodiment, inhibits (e.g., reduces, prevents)the expression of a PLVAP gene and/or gene product. Activities of aPLVAP protein that can be inhibited by an antagonist of the inventioninclude, but are not limited to, formation, growth, vascularizationand/or progression of a hepatocellular carcinoma tumor. In a particularembodiment, the PLVAP antagonist specifically binds a mammalian (e.g.,human) PLVAP protein and inhibits an activity of the PLVAP protein.

As used herein, “specifically binds” refers to binding of an agent(e.g., an antibody) to a PLVAP gene product (e.g., RNA, protein) with anaffinity (e.g., a binding affinity) that is at least about 5 fold,preferably at least about 10 fold, greater than the affinity with whichthe PLVAP antagonist binds a non-PLVAP protein.

As used herein, the term “polypeptide” refers to a polymer of aminoacids, and not to a specific length. Thus, “polypeptide” encompassesproteins, peptides, and oligopeptides.

As used herein, the term “antibody” refers to a polypeptide havingaffinity for a target, antigen, or epitope, and includes bothnaturally-occurring and engineered antibodies. The term “antibody”encompasses polyclonal, monoclonal, human, chimeric, humanized,primatized, veneered, and single chain antibodies, as well as fragmentsof antibodies (e.g., Fv, Fc, Fd, Fab, Fab′, F(ab′), scFv, scFab, dAb).(See, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold SpringHarbor Laboratory, 1988).

The term “antibody variable region” refers to the region of an antibodythat specifically binds an epitope (e.g., V_(H), V_(HH), V_(L)), eitherindependently or when combined with other antibody variable regions(e.g., a V_(H)/V_(L) pair).

The term “epitope” refers to a unit of structure conventionally bound byan antibody V_(H)/V_(L) pair. An epitope defines the minimum bindingsite for an antibody and, thus, represents the target of specificity ofan antibody.

The term “complementarity determining region,” or “CDR,” refers to ahypervariable region of an antibody variable region from a heavy chainor light chain that contains amino acid sequences capable ofspecifically binding to an antigenic target (e.g., epitope). A typicalheavy or light chain will have three CDRs (CDR1, CDR2, CDR3), whichaccount for the specificity of the antibody for a particular epitope.

As defined herein, the term “antigen binding fragment” refers to aportion of an antibody that contains one or more CDRs and has affinityfor an antigenic determinant by itself. Non-limiting examples includeFab fragments, F(ab)′₂ fragments, heavy-light chain dimers, and singlechain structures, such as a complete light chain or a complete heavychain.

As used herein, the term “specificity” refers to the ability of anantibody to bind preferentially to an epitope, and does not necessarilyimply high affinity.

The term “affinity” refers to a measure of the binding strength betweenan antibody and an antigenic determinant. Affinity depends on a numberof factors, including the closeness of stereochemical fit between theantibody and antigenic determinant, the size of the area of contactbetween them, and the distribution of charged and hydrophobic groups.

As used herein, the term “affinity constant,” or “K_(d),” refers to adissociation constant used to measure the affinity of an antibody for anantigen. The lower the affinity constant, the higher the affinity of theimmunoglobulin for the antigen or antigenic determinant, and vice versa.Such a constant is readily calculated from the rate constants for theassociation-dissociation reactions as measured by standard kineticmethodology for antibody reactions.

As referred to herein, the term “competes” means that the binding of afirst polypeptide (e.g., antibody) to a target antigen is inhibited bythe binding of a second polypeptide (e.g., antibody). For example,binding may be inhibited sterically, for example, by physical blockingof a binding domain or by alteration of the structure or environment ofa binding domain such that its affinity or avidity for a target isreduced.

As used herein, the term “peptide” refers to a compound consisting offrom about 2 to about 100 amino acid residues wherein the amino group ofone amino acid is linked to the carboxyl group of another amino acid bya peptide bond. Such peptides are typically less than about 100 aminoacid residues in length and preferably are about 10, about 20, about 30,about 40 or about 50 residues.

As used herein, the term “peptidomimetic” refers to molecules which arenot peptides or proteins, but which mimic aspects of their structures.Peptidomimetic antagonists can be prepared by conventional chemicalmethods (see, e.g., Damewood J. R. “Peptide Mimetic Design with the Aidof Computational Chemistry” in Reviews in Computational Biology, 2007,Vol. 9, pp. 1-80, John Wiley and Sons, Inc., New York, 1996; KazmierskiW. K., “Methods of Molecular Medicine: Peptidomimetic Protocols,” HumanaPress, New Jersey, 1999).

The terms “hepatocellular carcinoma,” “HCC,” and “hepatoma” are usedinterchangeably herein to refer to cancer that arises from hepatocytes,the major cell type of the liver.

As defined herein, “therapy” is the administration of a particulartherapeutic or prophylactic agent to a subject (e.g., a mammal, a human)that results in a desired therapeutic or prophylactic benefit to thesubject.

As defined herein, a “therapeutically effective amount” is an amountsufficient to achieve the desired therapeutic or prophylactic effectunder the conditions of administration, such as an amount sufficient toinhibit (i.e., reduce, prevent) tumor formation, tumor growth(proliferation, size), tumor vascularization and/or tumor progression(invasion, metastasis) in the liver of a patient with HCC. Theeffectiveness of a therapy (e.g., the reduction/elimination of a tumorand/or prevention of tumor growth) can be determined by any suitablemethod (e.g., in situ immunohistochemistry, imaging (ultrasound, CTscan, MRI, NMR), ³H-thymidine incorporation).

As defined herein, a “treatment regimen” is a regimen in which one ormore therapeutic or prophylactic agents are administered to a mammaliansubject at a particular dose (e.g., level, amount, quantity) and on aparticular schedule or at particular intervals (e.g., minutes, days,weeks, months).

As used herein, a “subject” refers to a mammalian subject. The term“mammalian subject” is defined herein to include mammals, such asprimates (e.g., humans), cows, sheep, goats, horses, dogs cats, rabbits,guinea pigs, rats, mice or other bovine, ovine, equine, canine feline,rodent or murine species. Examples of suitable subjects include, but arenot limited to, human patients who have, or are at risk for developing,HCC. Examples of high-risk groups for the development of HCC includeindividuals with chronic hepatitis infection (hepatitis B, hepatitis C)and individuals who have cirrhosis of the liver or related hepaticconditions.

The terms “prevent,” “preventing,” or “prevention,” as used herein, meanreducing the probability/likelihood or risk of HCC tumor formation orprogression by a subject, delaying the onset of a condition related toHCC in the subject, lessening the severity of one or more symptoms of anHCC-related condition in the subject, or any combination thereof. Ingeneral, the subject of a preventative regimen most likely will becategorized as being “at-risk,” e.g., the risk for the subjectdeveloping HCC is higher than the risk for an individual represented bythe relevant baseline population.

As used herein, the terms “treat,” “treating,” or “treatment” mean tocounteract a medical condition (e.g., a condition related to HCC) to theextent that the medical condition is improved according to aclinically-acceptable standard (e.g., reduced number and/or size of HCCtumors in a subject's liver).

As used herein, the terms “low stringency,” “medium stringency,” “highstringency,” and “very high stringency conditions” describe conditionsfor nucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated herein by reference in its entirety. Aqueous and nonaqueousmethods are described in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60 C; (3) high stringency hybridization conditionsin 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC,0.1% SDS at 65° C.; and preferably (4) very high stringencyhybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very highstringency conditions (4) are the preferred conditions and the ones thatshould be used unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4_(th)Ed, John Wiley & Sons, Inc., which are incorporated herein by reference)and chemical methods.

PLVAP

Plasmalemma vesicle-associated protein (PLVAP), also known as PV1, is atype II integral membrane glycoprotein whose expression is restricted tocertain vascular endothelial cells (Mol Biol Cell 15:3615-3630 (2004)).PLVAP has been shown to be a key structural component of fenestral andstomatal diaphragms of fenestrated endothelia. See id. In addition,PLVAP expression is necessary for the formation of endothelial fenestraldiaphragms and may be involved in modulating endothelial permeabilityand transport (Am J Physiol Heart Circ Physiol 286:H1347-1353, 2004).The genomic organization of human PLVAP gene has been reported (Stan RV, Arden K C, Palade G E. cDNA and protein sequence, genomicorganization, and analysis of cis regulatory elements of mouse and humanPLVAP genes. Genomics 72; 304-313, 2001).

As described herein, the inventors have demonstrated that PLVAP geneexpression is significantly elevated in hepatocellular carcinoma tissuesrelative to adjacent non-tumorous tissues in the liver of human HCCpatients. In addition, the present inventors have determined that PLVAPprotein is mainly expressed in, and localizes to, vascular endothelialcells surrounding or within HCC tumors, but is not expressed in, orlocalized to, cells associated with other liver pathologies.Accordingly, PLVAP represents a novel target for the diagnosis andtreatment of HCC.

Methods of Therapy

In one aspect, the invention relates to a method of treatinghepatocellular carcinoma (HCC) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of atleast one PLVAP antagonist, wherein the PLVAP antagonist inhibitsformation, growth, vascularization and/or progression of one or more HCCtumors in the liver of the subject. In a particular aspect, a PLVAPantagonist of the invention inhibits the expression or activity of PLVAPprotein in vascular endothelial cells surrounding hepatocytes in theliver of HCC patients.

In one aspect, a therapeutically-effective amount of a PLVAP antagonistis administered to a subject in need thereof to inhibit tumor growth orkill tumor cells. For example, agents which directly inhibit tumorgrowth (e.g., chemotherapeutic agents) are conventionally administeredat a particular dosing schedule and level to achieve the most effectivetherapy (e.g., to best kill tumor cells). Generally, about the maximumtolerated dose is administered during a relatively short treatmentperiod (e.g., one to several days), which is followed by an off-therapyperiod. In a particular example, the chemotherapeutic cyclophosphamideis administered at a maximum tolerated dose of 150 mg/kg every other dayfor three doses, with a second cycle given 21 days after the firstcycle. (Browder et al. Can Res 60:1878-1886, 2000).

A therapeutically-effective amount of PLVAP antagonist (e.g., inhibitorysmall molecules, neutralizing antibodies, inhibitory nucleic acids(e.g., siRNA, antisense nucleotides)) can be administered, for example,in a first cycle in which about the maximum tolerated dose of theantagonist is administered in one interval/dose, or in several closelyspaced intervals (minutes, hours, days) with another/second cycleadministered after a suitable off-therapy period (e.g., one or moreweeks). Suitable dosing schedules and amounts for a PLVAP antagonist canbe readily determined by a clinician of ordinary skill. Decreasedtoxicity of a particular PLVAP antagonist as compared tochemotherapeutic agents can allow for the time between administrationcycles to be shorter. When used as an adjuvant therapy (to, e.g.,surgery, radiation therapy, other primary therapies), atherapeutically-effective amount of a PLVAP antagonist is preferablyadministered on a dosing schedule that is similar to that of the othercancer therapy (e.g., chemotherapeutics), or on a dosing scheduledetermined by the skilled clinician to be more/most effective atinhibiting (reducing, preventing) tumor growth. A treatment regimen fora therapeutically-effective amount of an antibody PLVAP antagonist canbe, for example, from about 0.01 mg/kg to about 300 mg/kg body weightper treatment and preferably from about 0.01 mg/kg to about 100 mg/kg,from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10mg/kg every 1 to 7 days over a period of about 4 to about 6 months. Atreatment regimen for an anti-tumor effective amount of a small moleculePLVAP antagonist can be, for example, from about 0.001 mg/kg to about100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, every 1to 7 days over a period of about 4 to about 6 months.

In another aspect, a PLVAP antagonist can be administered in ametronomic dosing regime, whereby a lower dose is administered morefrequently relative to maximum tolerated dosing. A number of preclinicalstudies have demonstrated superior anti-tumor efficacy, potentantiangiogenic effects, and reduced toxicity and side effects (e.g.,myelosuppression) of metronomic regimes compared to maximum tolerateddose (MTD) counterparts (Bocci, et al., Cancer Res, 62:6938-6943,(2002); Bocci, et al., Proc. Natl. Acad. Sci., 100(22):12917-12922,(2003); and Bertolini, et al., Cancer Res, 63(15):4342-4346, (2003)).Metronomic chemotherapy appears to be effective in overcoming some ofthe shortcomings associated with chemotherapy.

A PLVAP antagonist can be administered in a metronomic dosing regime toinhibit (reduce, prevent) angiogenesis in a patient in need thereof aspart of an anti-angiogenic therapy. Such anti-angiogenic therapy mayindirectly affect (inhibit, reduce) tumor growth by blocking theformation of new blood vessels that supply tumors with nutrients neededto sustain tumor growth and enable tumors to metastasize. Starving thetumor of nutrients and blood supply in this manner can eventually causethe cells of the tumor to die by necrosis and/or apoptosis. Previouswork has indicated that the clinical outcomes (inhibition of endothelialcell-mediated tumor angiogenesis and tumor growth) of cancer therapiesthat involve the blocking of angiogenic factors (e.g., VEGF, bFGF,TGF-α, IL-8, PDGF) or their signaling have been more efficacious whenlower dosage levels are administered more frequently, providing acontinuous blood level of the antiangiogenic agent. (See Browder et al.Can. Res. 60:1878-1886, 2000; Folkman J., Sem. Can. Biol. 13:159-167,2003). An anti-angiogenic treatment regimen has been used with atargeted inhibitor of angiogenesis (thrombospondin 1 and platelet growthfactor-4 (TNP-470)) and the chemotherapeutic agent cyclophosphamide.Every 6 days, TNP-470 was administered at a dose lower than the maximumtolerated dose and cyclophosphamide was administered at a dose of 170mg/kg. See id. This treatment regimen resulted in complete regression ofthe tumors. See id. In fact, anti-angiogenic treatments are mosteffective when administered in concert with other anti-cancertherapeutic agents, for example, those agents that directly inhibittumor growth (e.g., chemotherapeutic agents). See id.

The therapeutic methods described herein comprise administering a PLVAPantagonist to a subject. The PLVAP antagonist may be administered to theindividual in need thereof as a primary therapy (e.g., as the principaltherapeutic agent in a therapy or treatment regimen); as an adjuncttherapy (e.g., as a therapeutic agent used together with anothertherapeutic agent in a therapy or treatment regime, wherein thecombination of therapeutic agents provides the desired treatment;“adjunct therapy” is also referred to as “adjunctive therapy”); incombination with an adjunct therapy; as an adjuvant therapy (e.g., as atherapeutic agent that is given to the subject in need thereof after theprincipal therapeutic agent in a therapy or treatment regimen has beengiven); or in combination with an adjuvant therapy (e.g., chemotherapy(e.g., tamoxifen, cisplatin, mitomycin, 5-fluorouracil, doxorubicin,sorafenib, octreotide, dacarbazine (DTIC), Cis-platinum, cimetidine,cyclophophamide), radiation therapy (e.g., proton beam therapy), hormonetherapy (e.g., anti-estrogen therapy, androgen deprivation therapy(ADT), luteinizing hormone-releasing hormone (LH-RH) agonists, aromataseinhibitors (AIs, such as anastrozole, exemestane, letrozole), estrogenreceptor modulators (e.g., tamoxifen, raloxifene, toremifene)), orbiological therapy). Numerous other therapies can also be administeredduring a cancer treatment regime to mitigate the effects of the diseaseand/or side effects of the cancer treatment, including therapies tomanage pain (narcotics, acupuncture), gastric discomfort (antacids),dizziness (anti-vertigo medications), nausea (anti-nausea medications),infection (e.g., medications to increase red/white blood cell counts)and the like, all of which are readily appreciated by the person skilledin the art.

Thus, a PLVAP antagonist can be administered as an adjuvant therapy(e.g., with another primary cancer therapy or treatment). As an adjuvanttherapy, the PLVAP antagonist can be administered before, after orconcurrently with a primary therapy like radiation and/or the surgicalremoval of a tumor(s). In some embodiments, the method comprisesadministering a therapeutically effective amount of a PLVAP antagonistand one or more other therapies (e.g., adjuvant therapies, othertargeted therapies). An adjuvant therapy (e.g., a chemotherapeuticagent) and/or the one or more other targeted HCC therapies and the PLVAPantagonist can be co-administered simultaneously (e.g., concurrently)either as separate formulations or as a joint formulation.Alternatively, the therapies can be administered sequentially, asseparate compositions, within an appropriate time frame (e.g., a cancertreatment session/interval such as 1.5 to 5 hours) as determined by theskilled clinician (e.g., a time sufficient to allow an overlap of thepharmaceutical effects of the therapies). The adjuvant therapy and/orone or more other targeted HCC therapies and the PLVAP antagonist can beadministered in a single dose or multiple doses in an order and on aschedule suitable to achieve a desired therapeutic effect (e.g.,inhibition of tumor growth, inhibition of angiogenesis, and/orinhibition of cancer metastasis).

One or more agents that are PLVAP antagonists can be administered insingle or multiple doses. Suitable dosing and regimens of administrationcan be determined by a clinician and are dependent on the agent(s)chosen, pharmaceutical formulation and route of administration, variouspatient factors and other considerations. With respect to theadministration of a PLVAP antagonist with one or more other therapies ortreatments (adjuvant, targeted, cancer treatment-associated, and thelike) the PLVAP antagonist is typically administered as a single dose(e.g., by injection, by infusion, orally), followed by repeated doses atparticular intervals (e.g., one or more hours) if desired or indicated.

The amount of the PLVAP antagonist to be administered (e.g., atherapeutically effective amount) can be determined by a clinician usingthe guidance provided herein and other methods known in the art and isdependent on several factors, including, for example, the particularagent chosen, the subject's age, sensitivity, tolerance to drugs andoverall well-being. For example, suitable dosages for a small moleculecan be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kgto about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about0.01 mg/kg to about 1 mg/kg body weight per treatment. Suitable dosagesfor antibodies can be from about 0.01 mg/kg to about 300 mg/kg bodyweight per treatment and preferably from about 0.01 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg toabout 10 mg/kg body weight per treatment. Where the PLVAP antagonist isa polypeptide (linear, cyclic, mimetic), the preferred dosage willresult in a plasma concentration of the peptide from about 0.1 μg/mL toabout 200 μg/mL. Determining the dosage for a particular agent, patientand cancer is well within the abilities of one of skill in the art.Preferably, the dosage does not cause or produces minimal adverse sideeffects (e.g., immunogenic response, nausea, dizziness, gastric upset,hyperviscosity syndromes, congestive heart failure, stroke, pulmonaryedema).

Methods for Administration

According to the methods of the invention, a therapeutically effectiveamount of a PLVAP antagonist (e.g., antibody, such as an antibodylabeled with a radioactive isotope) is administered to a mammaliansubject to treat HCC.

A variety of routes of administration can be used, including, forexample, oral, dietary, topical, transdermal, rectal, parenteral (e.g.,intraaterial, intravenous, intramuscular, subcutaneous injection,intradermal injection), intravenous infusion and inhalation (e.g.,intrabronchial, intranasal or oral inhalation, intranasal drops) routesof administration, depending on the agent and the particular cancer tobe treated. Administration can be local or systemic as indicated. Thepreferred mode of administration can vary depending on the particularagent chosen; however, intraarterial administration (e.g., hepaticarterial infusion, trans-arterial chemoembolization (TACE)) is generallypreferred to administer therapeutic agents (e.g., antibodies, such asantibodies labeled with a radioactive isotope) of the invention to treathepatocellular carcinoma.

For example, using hepatic arterial infusion, chemotherapeutic agents(e.g., PLVAP antibodies, such as PLVAP antibodies labeled with aradioactive isotope) can be delivered directly to an HCC tumor throughthe hepatic artery, for example, during routine TACE treatment of HCC(Camma, et al. Radiology 224:47-54, 2002; Befeler, et al. Clinics inLiver Disease 9:287-300, 2005; Abou-Alfa JAMA 299:1716-1718, 2008). Thisprocedure is done with the help of fluoroscopy (type of x-ray) imaging.Briefly, a catheter is inserted into the femoral artery in the groin andis threaded into the aorta. From the aorta, the catheter is advancedinto the hepatic artery or its branches. Once the branches of thehepatic artery that feed the liver cancer are identified, thechemotherapy is infused. An interventional radiologist, who usuallycarries out this procedure, can determine the amount of chemotherapythat a patient receives at each session. Some patients may undergorepeat sessions at 6 to 12 week intervals. Imaging studies of the liverare repeated in six to 12 weeks to assess the size of the tumor inresponse to the treatment.

Alternatively, trans-arterial chemoembolization (TACE), a procedure thatis similar to intraarterial infusion, can be used to administer PLVAPantagonists (e.g., antibodies) to a subject in need thereof. In TACE,intraarterial infusion of a therapeutic agent is combined with theadditional step of blocking (i.e., embolizing) the small blood vesselswith particular blocking compounds, such as gelfoam, oil emulsion, oreven small metal coils. Thus, TACE has the potential advantages ofexposing the tumor to high concentrations of chemotherapy and confiningthe agents locally in order to prevent or reduce their being carriedaway by the blood stream. At the same time, TACE deprives the tumor ofits needed blood supply, which can result in the damage or death of thetumor cells.

For intraarterial administration of PLVAP antibodies, it is preferred touse antibodies having high affinities to PLVAP (e.g., a K_(d) less than10⁻⁷ M) so that the infused antibodies will be concentrated in bloodvessels of HCC. Chimeric and humanized antibodies are expected to havecirculatory half-lives of up to four and up to 14-21 days, respectively.In a particular embodiment, high affinity PLVAP antibodies (e.g.,antigen binding fragments, single chain antibodies) with shortcirculatory half-lives (e.g., about 1 day to about 5 days, for example,about 1, 2, 3, 4 or 5 days) are administered to a patient in order toreduce any toxicity and other adverse side-effects resulting from theiradministration. In another embodiment, high affinity PLVAP antibodieswith long circulatory half-lives (e.g., about 5 days to about 24 days)are administered to a patient to treat HCC.

In many cases, it will be preferable to administer a large loading dosefollowed by periodic (e.g., weekly) maintenance doses over the treatmentperiod. Antibodies can also be delivered by slow-release deliverysystems, pumps, and other known delivery systems for continuous infusioninto HCC. Dosing regimens may be varied to provide the desiredcirculating levels of a particular antibody based on itspharmacokinetics. Thus, doses will be calculated so that the desiredtherapeutic level is maintained.

The actual dose and treatment regimen will be determined by thephysician, taking into account the nature of the cancer (primary ormetastatic), number and size of tumors, other therapies, and patientcharacteristics. In view of the life-threatening nature ofhepatocellular carcinoma, large doses with significant side effects maybe employed.

Nucleic acid-based PLVAP antagonists (e.g., siRNAs, antisenseoligonucleotides, natural or synthetic nucleic acids, nucleic acidanalogs) can be introduced into a mammalian subject of interest in anumber of ways. For instance, nucleic acids may be expressedendogenously from expression vectors or PCR products in host cells orpackaged into synthetic or engineered compositions (e.g., liposomes,polymers, nanoparticles) that can then be introduced directly into thebloodstream of a mammalian subject (by, e.g., injection, infusion).Anti-PLVAP nucleic acids or nucleic acid expression vectors (e.g.,retroviral, adenoviral, adeno-associated and herpes simplex viralvectors, engineered vectors, non-viral-mediated vectors) can also beintroduced into a mammalian subject directly using established genetherapy strategies and protocols (see, e.g., Tochilin V. P. Annu RevBiomed Eng 8:343-375, 2006; Recombinant DNA and Gene Transfer, Office ofBiotechnology Activities, National Institutes of Health Guidelines).

Similarly, where the agent is a protein or polypeptide, the agent can beadministered via in vivo expression of recombinant protein. In vivoexpression can be accomplished by somatic cell expression according tosuitable methods (see, e.g., U.S. Pat. No. 5,399,346). Further, anucleic acid encoding the polypeptide can also be incorporated intoretroviral, adenoviral or other suitable vectors (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the polypeptide for delivery. In the latter embodiment, thecells can be implanted (alone or in a barrier device), injected orotherwise introduced in an amount effective to express the polypeptidein a therapeutically effective amount.

Diagnostic and Prognostic Methods

The present invention encompasses diagnostic and prognostic methods thatcomprise assessing expression of PLVAP in a sample (e.g., liver biopsy,fine needle aspiration sample) from a mammalian subject (e.g., amammalian subject who has a liver tumor). For diagnostic methods of theinvention, expression of PLVAP in the sample, or increased expression ofPLVAP in the sample relative to a suitable control, indicates that thesubject has HCC, and/or that the subject is a candidate for ananti-cancer therapy using a PLVAP antagonist.

For prognostic methods of the invention, expression of PLVAP in a samplefrom a subject, or increased expression of PLVAP in the sample relativeto a suitable control, indicates a poor prognosis. The prognosis can bea prognosis for patient survival, a prognosis for risk of metastasesand/or a prognosis for risk of relapse.

Suitable samples for these methods include a tissue sample, a biologicalfluid sample, a cell(s) (e.g., a tumor cell) sample, and the like. Anymeans of sampling from a subject, for example, by blood draw, spinaltap, tissue smear or scrape, or tissue biopsy, can be used to obtain asample. Thus, the sample can be a biopsy specimen (e.g., tumor, polyp,mass (solid, cell)), aspirate, smear or blood sample. The sample can bea tissue from a liver that has a tumor (e.g., cancerous growth) and/ortumor cells, or is suspected of having a tumor and/or tumor cells. Forexample, a tumor biopsy can be obtained in an open biopsy, a procedurein which an entire (excisional biopsy) or partial (incisional biopsy)mass is removed from a target area. Alternatively, a tumor sample can beobtained through a percutaneous biopsy, a procedure performed with aneedle-like instrument through a small incision or puncture (with orwithout the aid of a imaging device) to obtain individual cells orclusters of cells (e.g., a fine needle aspiration (FNA)) or a core orfragment of tissues (core biopsy). The biopsy samples can be examinedcytologically (e.g., smear), histologically (e.g., frozen or paraffinsection) or using any other suitable method (e.g., molecular diagnosticmethods). A tumor sample can also be obtained by in vitro harvest ofcultured human cells derived from an individual's tissue. Tumor samplescan, if desired, be stored before analysis by suitable storage meansthat preserve a sample's protein and/or nucleic acid in an analyzablecondition, such as quick freezing, or a controlled freezing regime. Ifdesired, freezing can be performed in the presence of a cryoprotectant,for example, dimethyl sulfoxide (DMSO), glycerol, orpropanediol-sucrose. Tumor samples can be pooled, as appropriate, beforeor after storage for purposes of analysis. The tumor sample can be froma patient who has a liver cancer, for example, hepatocellular carcinoma.

Suitable assays that can be used to assess the presence or amount of aPLVAP in a sample (e.g., biological sample) are known to those of skillin the art. Methods to detect a PLVAP protein or peptide includeimmunological and immunochemical methods like flow cytometry (e.g., FACSanalysis), enzyme-linked immunosorbent assays (ELISA), includingchemiluminescence assays, radioimmunoassay, immunoblot (e.g., Westernblot), immunohistochemistry (IHC), and other antibody-based quantitativemethods (e.g., Luminex® beads-based assays). Other suitable methodsinclude, for example, mass spectroscopy. For example, antibodies toPLVAP can be used to determine the presence and/or expression level ofPLVAP in a sample directly or indirectly using, e.g.,immunohistochemistry (IHC). For instance, paraffin sections can be takenfrom a biopsy, fixed to a slide and combined with one or more antibodiesby suitable methods. In a particular embodiment, detection of PLVAPprotein in vascular endothelial cells surrounding hepatocytes in asample is indicative of HCC.

An exemplary ELISA assay for use in diagnostic and prognosticapplications of the invention is described in Example 9 herein.

Methods to detect PLVAP gene expression include PLVAP nucleic acidamplification and/or visualization. To detect PLVAP gene expression, anucleic acid can be isolated from an individual by suitable methodswhich are routine in the art (see, e.g., Sambrook et al., 1989).Isolated nucleic acid can then be amplified (by, e.g., polymerase chainreaction (PCR) (e.g., direct PCR, quantitative real time PCR, reversetranscriptase PCR), ligase chain reaction, self sustained sequencereplication, transcriptional amplification system, Q-Beta Replicase, orthe like) and visualized (by, e.g., labeling of the nucleic acid duringamplification, exposure to intercalating compounds/dyes, probes). PLVAPRNA (e.g., mRNA) or expression thereof can also be detected using anucleic acid probe, for example, a labeled nucleic acid probe (e.g.,fluorescence in situ hybridization (FISH)) directly in a paraffinsection of a tissue sample taken from, e.g., a tumor biopsy, or usingother suitable methods. PLVAP gene expression thereof can also beassessed by Southern blot or in solution (e.g., dyes, probes). Further,a gene chip, microarray, probe (e.g., quantum dots) or other such device(e.g., sensor, nanonsensor/detector) can be used to detect expressionand/or differential expression of a PLVAP gene.

In one embodiment, a hepatocellular carcinoma can be diagnosed bydetecting expression of a PLVAP gene product (e.g., PLVAP mRNA, PLVAPprotein) in a sample from a patient. Thus, the method does not requirethat PLVAP expression in the sample from the patient be compared to theexpression of PLVAP in a control. The presence or absence of PLVAP canbe ascertained by the methods described herein or other suitable assays.In another embodiment, an increase in expression of PLVAP can bedetermined by comparison of PLVAP expression in the sample to that of asuitable control. Suitable controls include, for instance, anon-neoplastic tissue sample from the individual, non-cancerous cells,non-metastatic cancer cells, non-malignant (benign) cells or the like,or a suitable known or determined reference standard. The referencestandard can be a typical, normal or normalized range or level ofexpression of a PLVAP protein or RNA (e.g., an expression standard).Thus, the method does not require that expression of the gene/protein beassessed in a suitable control.

In another embodiment, a hepatocellular carcinoma can be diagnosed bydetecting the PLVAP gene copy number in a sample from a patient. Forexample, in some embodiments, a PLVAP gene copy number that is greaterthan two (e.g., a gene copy number of 3 or 4) can be diagnostic of HCC.Typically, a normal human cell will have a PLVAP gene copy number oftwo. Therefore, a method of diagnosis based on PLVAP gene copy numberdoes not require detecting the PLVAP gene copy number in a controlsample from the patient, although a control may be used. Suitablecontrols include, for instance, a non-neoplastic tissue sample from theindividual, non-cancerous cells, non-metastatic cancer cells,non-malignant (benign) cells or the like, or a suitable known ordetermined reference standard (e.g., a PLVAP gene copy number of two).The copy number of the PLVAP gene in a sample from a patient can beascertained by suitable techniques, such as, for example, fluorescencein situ hybridization (FISH).

PLVAP Antibodies

As described herein, antibodies that bind PLVAP have utility in thediagnosis and treatment of HCC in human subjects. For example,antibodies that specifically bind PLVAP can be used to detect thepresence of PLVAP on capillary endothelial cells of hepatocellularcarcinoma in specimens of liver core biopsies or needle aspirates byimmunohistochemical staining (IHC). In addition, antibodies (e.g.,humanized antibodies, chimeric antibodies) to PLVAP can be labeled witha proper tracer (e.g., radioisotope) for immuno-positron emissiontomography (immuno-PET) (Clin Cancer Res 12:1958-1960, 2006; Clin CancerRes 12:2133-2140, 2006) to determine whether a space occupying lesion(s)in the liver of a subject is hepatocellular carcinoma. Anti-PLVAPantibodies (e.g., humanized antibodies) can also be labeled with acytotoxic agent (radioactive or non-radioactive) for therapeuticpurposes (Weiner L M, Adams G P, Von Mehren M. Therapeutic monoclonalantibodies: General principles. In: Cancer: Principles & Practice ofOncology. 6^(th) ed. DeVita V T, Hellman S, Rosenberg S A, eds.Philadelphia: Lippincott Williams & Wilkins; 2001:495-508.; Levinson W,Jawetz E. Medical Microbiology & Immunology. 4^(th) ed. Stamford:Appleton & Lange; 1996:307-47; Scheinberg D A, Sgouros G, Junghans R P.Antibody-based immunotherapies for cancer. In: Cancer Chemotherapy &Biotherapy: Principles and Practice. 3^(rd) ed. Chabner B A, Longo D L,eds. Philadelphia: Lippincott Williams & Wilkins; 2001:850-82).

Accordingly, in one embodiment, the invention provides an antibody thatbinds (e.g., specifically binds) a PLVAP protein (e.g., a human PLVAPprotein (SEQ ID NO:23)). Antibodies that specifically bind to a PLVAPprotein can be polyclonal, monoclonal, human, chimeric, humanized,primatized, veneered, and single chain antibodies, as well as fragmentsof antibodies (e.g., Fv, Fc, Fd, Fab, Fab′, F(ab′), scFv, scFab, dAb),among others. (See, e.g., Harlow et al., Antibodies A Laboratory Manual,Cold Spring Harbor Laboratory, 1988). Antibodies that specifically bindto a PLVAP protein can be produced, constructed, engineered and/orisolated by conventional methods or other suitable techniques. Forexample, antibodies which are specific for a PLVAP protein can be raisedagainst an appropriate immunogen, such as a recombinant mammalian (e.g.,human) PLVAP protein (e.g., SEQ ID NO:23) or a portion thereof (e.g.,SEQ ID NO:2, SEQ ID NO:38, SEQ ID NO:40) (including synthetic molecules,e.g., synthetic peptides). A variety of such immunization methods havebeen described (see, e.g., Kohler et al., Nature, 256: 495-497 (1975)and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E.and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Antibodies can also be raised by immunizing a suitable host (e.g.,mouse) with cells that express PLVAP (e.g., cancer cells/cell lines) orcells engineered to express PLVAP (e.g., transfected cells). (See, e.g.,Chuntharapai et al., J. Immunol., 152:1783-1789 (1994); Chuntharapai etal. U.S. Pat. No. 5,440,021).

At an appropriate time after immunization, e.g., when the antibodytiters are highest, antibody-producing cells can be obtained from theimmunized animal and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (Nature 256:495-497, 1975), the human B cellhybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96, 1985) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc.,New York, N.Y., 1994). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds a polypeptidedescribed herein.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the invention (see, e.g.,Current Protocols in Immunology, supra; Galfre et al., Nature,266:55052, 1977; R. H. Kenneth, in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y., 1980; and Lerner, Yale J. Biol. Med. 54:387-402, 1981). Moreover,the ordinarily skilled worker will appreciate that there are manyvariations of such methods that also would be useful.

In one embodiment, the invention relates to a monoclonal anti-PLVAPantibody produced by murine hybridoma KFCC-GY4 (ATCC Patent DepositDesignation PTA-9963), having been deposited on Apr. 8, 2009, at theAmerican Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va.20108, United States of America. In another embodiment, the inventionrelates to a monoclonal anti-PLVAP antibody produced by murine hybridomaKFCC-GY5 (ATCC Patent Deposit Designation PTA-9964), having beendeposited on Apr. 8, 2009, at the American Type Culture Collection(ATCC), P.O. Box 1549, Manassas, Va. 20108, United States of America.The invention further relates to the murine hybridoma cell linesKFCC-GY4 (ATCC Patent Deposit Designation PTA-9963) and KFCC-GY5 (ATCCPatent Deposit Designation PTA-9964) themselves, as well as cellsobtained from these hybridomas.

In one alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal antibody to a PLVAP protein can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with the targetpolypeptide to thereby isolate immunoglobulin library members that bindthe polypeptide. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examplesof methods and reagents particularly amenable for use in generating andscreening antibody display libraries can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al.,Hum. Antibodies Hybridomas 3:81-85, 1992; Huse et al., Science246:1275-1281, 1989; and Griffiths et al., EMBO J. 12:725-734, 1993.

Antibody fragments (e.g., antigen-binding fragments) can be produced byenzymatic cleavage or by recombinant techniques. For example, papain orpepsin cleavage can generate Fab or F(ab′)₂ fragments, respectively.Other proteases with the requisite substrate specificity can also beused to generate Fab or F(ab′)₂ fragments.

Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons has been introducedupstream of the natural stop site. For example, a chimeric gene encodinga F(ab′)₂ heavy chain portion can be designed to include DNA sequencesencoding the CH₁ domain and hinge region of the heavy chain.

Single chain, human, chimeric, humanized, primatized (CDR-grafted), orveneered antibodies comprising portions derived from different speciesare also encompassed by the present invention and the term “antibody.”The various portions of these antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; Queen et al., European PatentNo. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596 A1. See alsoNewman, R. et al., BioTechnology, 10: 1455-1460 (1992) regardingprimatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 andBird, R. E. et al., Science, 242: 423-426 (1988)) regarding single chainantibodies.

In a particular embodiment, the invention relates to chimeric antibodiesthat specifically bind to PLVAP (e.g., a human PLVAP protein comprisingSEQ ID NO:23). In one embodiment, chimeric antibody of the inventioncomprises at least one heavy chain and at least one light chain (e.g.,kappa light chain) of human IgG4. The production and characterization ofexemplary chimeric antibodies of the invention are described in Example7 herein.

In another embodiment, the invention relates to humanized antibodiesthat specifically bind to PLVAP (e.g., a human PLVAP protein comprisingSEQ ID NO:23). Humanized antibodies of the invention can comprise, forexample, at least one heavy chain amino acid sequence selected from thegroup consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102 and a combination thereof and/or at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108and a combination thereof.

Humanized antibodies can be produced using synthetic or recombinant DNAtechnology using standard methods or other suitable techniques. Nucleicacid (e.g., cDNA) sequences coding for humanized variable regions canalso be constructed using PCR mutagenesis methods to alter DNA sequencesencoding a human or humanized chain, such as a DNA template from apreviously humanized variable region (see, e.g., Kamman, M., et al.,Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research,53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302(1991)). Using these or other suitable methods, variants can also bereadily produced. In one embodiment, cloned variable regions (e.g.,dAbs) can be mutated, and sequences encoding variants with the desiredspecificity can be selected (e.g., from a phage library; see, e.g.,Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213,published Apr. 1, 1993).

Humanized antibodies can also be produced by and/or obtained fromcommercial sources, including, for example, Antitope Limited (Cambridge,UK). An exemplary method of producing humanized antibodies that is basedon the Composite Human Antibody™ technology of Antitope Limited isdescribed in Example 8 herein.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, for example, methods whichselect a recombinant antibody or antibody-binding fragment (e.g., dAbs)from a library (e.g., a phage display library), or which rely uponimmunization of transgenic animals (e.g., mice). Transgenic animalscapable of producing a repertoire of human antibodies are well-known inthe art (e.g., Xenomouse® (Abgenix, Fremont, Calif.)) and can beproduced using suitable methods (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807; Lonberg et al., WO 97/13852).

Once produced, an antibody specific for PLVAP can be readily identifiedusing methods for screening and isolating specific antibodies that arewell known in the art. See, for example, Paul (ed.), FundamentalImmunology, Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43:1-98,1988; Goding (ed.), Monoclonal Antibodies: Principles and Practice,Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:67-101,1984. A variety of assays can be utilized to detect antibodies thatspecifically bind to PLVAP proteins. Exemplary assays are described indetail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), ColdSpring Harbor Laboratory Press, 1988. Representative examples of suchassays include: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assays, inhibition or competition assays, andsandwich assays.

In certain embodiments, the antibodies of the invention have a highbinding affinity for PLVAP. Such antibodies will preferably have anaffinity (e.g., binding affinity) for PLVAP, expressed as K_(d), of atleast about 10⁻⁷ M (e.g., about 0.4×10⁻⁷ M, about 0.6×10⁻⁷ M, about4.06×10⁻⁷ M, about 4.64×10⁻⁷ M), or higher, for example, at least about10⁻⁸ M (e.g., about 5.98×10⁻⁸ M), at least about 10⁻⁹ M, or at leastabout 10⁻¹⁰ M (e.g., about 9.78×10⁻¹⁰ M), such as about 9.78×10⁻¹⁰ M.The binding affinity of an antibody can be readily determined by one ofordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949). Binding affinitycan also be determined using a commercially available biosensorinstrument (BIACORE, Pharmacia Biosensor, Piscataway, N.J.), whereinprotein is immobilized onto the surface of a receptor chip. SeeKarlsson, J. Immunol. Methods 145:229-240, 1991 and Cunningham andWells, J. Mol. Biol. 234:554-563, 1993. This system allows thedetermination of on- and off-rates, from which binding affinity can becalculated, and assessment of stoichiometry of binding.

The antibodies of the present invention can include a label, such as,for example, a detectable label that permits detection of the antibody,and proteins bound by the antibody (e.g., PLVAP), in a biologicalsample. A detectable label is particularly suitable for diagnosticapplications. For example, a PLVAP antibody can be labeled with aradioactive isotope (radioisotope), which can be detected by one ofskill in the art using a gamma counter or a scintillation counter or byautoradiography or other suitable means. Isotopes which are useful forthe purpose of the present invention include, but are not limited to:³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe and ⁷⁵Se.

Antibodies of the invention can also be labeled with a fluorescentcompound (e.g., dyes). When the fluorescently labeled antibody isexposed to light of the proper wavelength, its presence can then bedetected due to the fluorescence of the compound. Among the mostcommonly used fluorescent labels are fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine. The antibodies of the invention can also be labeledusing fluorescence emitting metals, such as ¹⁵²Eu or others of thelanthanide series. These metals can be attached to the antibody moleculeusing such metal chelating groups as diethylenetriaminepentaacetic acid(DTPA), tetraaza-cyclododecane-tetraacetic acid (DOTA) orethylenediaminetetraacetic acid (EDTA).

The antibodies of the present invention also can be coupled to achemiluminescent compound. Examples of useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Useful bioluminescent compounds for purposes of labelingantibodies are luciferin, luciferase and aequorin.

Detection of the labeled antibodies can be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorimetric methods that employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of the enzymatic reaction of a substrate tosimilarly prepared standards.

Accordingly, the antibodies of the present invention can also be used asa stain for tissue sections. For example, a labeled antibody that bindsto PLVAP can be contacted with a tissue sample, e.g., a liver tissuebiopsy or fine needle aspirate from a patient. This section may then bewashed and the label detected using an appropriate means.

For the purpose of treating HCC, PLVAP antibodies of the invention mayinclude a radiolabel or other therapeutic agent that enhancesdestruction of cells expressing PLVAP (e.g., vascular endothelial cellssurrounding HCC cells). Examples of suitable radioisotope labels for usein HCC therapy include, but are not limited to, ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu,²¹⁷Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ¹¹¹In and ¹¹⁸Re. Optionally, a labelthat emits α and β particles upon bombardment with neutron radiation,such as boron, can be used as a label for therapeutic PLVAP antibodies.

Therapeutic antibodies also may include a cytotoxic agent that iscapable of selectively killing cells that express PLVAP. For example,bacterial toxins, such as diphtheria toxin or ricin, can be used.Methods for producing antibodies comprising fragment A of diphtheriatoxin are taught in U.S. Pat. No. 4,675,382 (1987). Diphtheria toxincontains two polypeptide chains. The B chain binds the toxin to areceptor on a cell surface. The A chain actually enters the cytoplasmand inhibits protein synthesis by inactivating elongation factor 2, thefactor that translocates ribosomes along mRNA concomitant withhydrolysis of ETP. See Darnell, J. et al., in Molecular Cell Biology,Scientific American Books, Inc., page 662 (1986). Alternatively, anantibody comprising ricin, a toxic lectin, may be prepared. Othersuitable cytotoxic agents are known by those of skill in the art.

For in vivo detection, PLVAP antibodies of the invention may beconjugated to radionuclides either directly or by using an intermediaryfunctional group. An intermediary group which is often used to bindradioisotopes, which exist as metallic cations, to antibodies isdiethylenetriaminepentaacetic acid (DTPA) ortetraaza-cyclododecane-tetraacetic acid (DOTA). Typical examples ofmetallic cations which are bound in this manner are ⁹⁹Tc, ¹²³I, ¹¹¹In,¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga.

Moreover, the antibodies of the invention may be tagged with an NMRimaging agent that includes paramagnetic atoms. The use of an NMRimaging agent allows the in vivo diagnosis of the presence of and theextent of HCC in a patient using NMR techniques. Elements which areparticularly useful in this manner are ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe.

PLVAP Antagonists

A PLVAP antagonist of the invention can be any agent that inhibits(e.g., reduces, prevents) an activity of a PLVAP gene product. PLVAPactivities include, but are not limited to, formation, growth,vascularization or progression of an HCC tumor. In a particularembodiment, a PLVAP antagonist inhibits an activity of a PLVAP geneproduct (e.g., PLVAP RNA, PLVAP protein) by specifically binding to thePLVAP gene product. PLVAP antagonists also encompass agents that inhibit(reduce, decrease, prevent) the expression (e.g., transcription, mRNAprocessing, translation) of a PLVAP gene or gene product (e.g., PLVAPRNA, PLVAP protein). A PLVAP antagonist can be an antibody, a smallmolecule, a peptide, a peptidomimetic, or a nucleic acid, among others.

Antibody Antagonists

A PLVAP antagonist of the invention can be an antibody that specificallybinds a PLVAP protein. Such antibodies include, but are not limited to,any of the PLVAP-specific antibodies described herein.

Small Molecule Antagonists

PLVAP antagonists can also be small molecules. Examples of smallmolecules include organic compounds, organometallic compounds, inorganiccompounds, and salts of organic, organometallic or inorganic compounds.Atoms in a small molecule are typically linked together via covalentand/or ionic bonds. The arrangement of atoms in a small organic moleculemay represent a chain (e.g., a carbon-carbon chain or acarbon-heteroatom chain), or may represent a ring containing carbonatoms, e.g., benzene or a policyclic system, or a combination of carbonand heteroatoms, i.e., heterocycles such as a pyrimidine or quinazoline.Although small molecules can have any molecular weight, they generallyinclude molecules that are less than about 5,000 daltons. For example,such small molecules can be less than about 1000 daltons and,preferably, are less than about 750 daltons or, more preferably, areless than about 500 daltons. Small molecules and other non-peptidicPLVAP antagonists can be found in nature (e.g., identified, isolated,purified) and/or produced synthetically (e.g., by traditional organicsynthesis, bio-mediated synthesis, or a combination thereof). See, e.g.,Ganesan, Drug Discov. Today 7(1): 47-55 (January 2002); Lou, DrugDiscov. Today, 6(24): 1288-1294 (December 2001). Examples of naturallyoccurring small molecules include, but are not limited to, hormones,neurotransmitters, nucleotides, amino acids, sugars, lipids, and theirderivatives.

Peptide Antagonists

The PLVAP antagonist of the invention can also be a peptide that bindsto a PLVAP protein. The peptide can comprise any suitable L- and/orD-amino acid, for example, common α-amino acids (e.g., alanine, glycine,valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid,6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g.,citrulline, homocitruline, homoserine, norleucine, norvaline,ornithine). The amino, carboxyl and/or other functional groups on apeptide can be free (e.g., unmodified) or protected with a suitableprotecting group. Suitable protecting groups for amino and carboxylgroups and methods for adding or removing protecting groups are known inthe art and are disclosed in, for example, Green and Wuts, “ProtectingGroups in Organic Synthesis,” John Wiley and Sons, 1991. The functionalgroups of a peptide can also be derivatized (e.g., alkylated) usingart-known methods.

The peptide PLVAP antagonist can comprise one or more modifications(e.g., amino acid linkers, acylation, acetylation, amidation,methylation, terminal modifiers (e.g., cyclizing modifications)), ifdesired. The peptide can also contain chemical modifications (e.g.,N-methyl-α-amino group substitution). In addition, the peptideantagonist can be an analog of a known and/or naturally-occurringpeptide, for example, a peptide analog having conservative amino acidresidue substitution(s). These modifications can improve variousproperties of the peptide (e.g., solubility, binding), including itsPLVAP antagonist activity.

PLVAP antagonists that are peptides can be linear, branched or cyclic,e.g., a peptide having a heteroatom ring structure that includes severalamide bonds. In a particular embodiment, the peptide is a cyclicpeptide. Such peptides can be produced by one of skill in the art usingstandard techniques. For example, a peptide can be derived or removedfrom a native protein by enzymatic or chemical cleavage, or can besynthesized by suitable methods, for example, solid phase peptidesynthesis (e.g., Merrifield-type synthesis) (see, e.g., Bodanszky et al.“Peptide Synthesis,” John Wiley & Sons, Second Edition, 1976). Peptidesthat are PLVAP antagonists can also be produced, for example, usingrecombinant DNA methodologies or other suitable methods (see, e.g.,Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001).

Peptides can be synthesized and assembled into libraries comprising afew to many discrete molecular species. Such libraries can be preparedusing methods of combinatorial chemistry, and can be screened using anysuitable method to determine if the library comprises peptides with adesired biological activity. Such peptide antagonists can then beisolated using suitable methods known by those of skill in the art.

Peptidomimetic Antagonists

PLVAP antagonists can also be peptidomimetics. For example,polysaccharides can be prepared that have the same functional groups aspeptides. Peptidomimetics can be designed, for example, by establishingthe three dimensional structure of a peptide agent in the environment inwhich it is bound or will bind to a target molecule. The peptidomimeticcomprises at least two components, the binding moiety or moieties andthe backbone or supporting structure.

The binding moieties are the chemical atoms or groups which will reactor form a complex (e.g., through hydrophobic or ionic interactions) witha target molecule, for instance, human PLVAP. For example, the bindingmoieties in a peptidomimetic can be the same as those in a peptide orprotein antagonist. The binding moieties can be an atom or chemicalgroup which reacts with the receptor in the same or similar manner asthe binding moiety in the peptide antagonist. For example, computationalchemistry can be used to design peptide mimetics of peptides that bindPLVAP proteins. Examples of binding moieties suitable for use indesigning a peptidomimetic for a basic amino acid in a peptide includenitrogen containing groups, such as amines, ammoniums, guanidines andamides or phosphoniums. Examples of binding moieties suitable for use indesigning a peptidomimetic for an acidic amino acid include, forexample, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, alower alkyl sulfonic acid ester or a phosphorous acid or ester thereof.

The supporting structure is the chemical entity that, when bound to thebinding moiety or moieties, provides the three dimensional configurationof the peptidomimetic. The supporting structure can be organic orinorganic. Examples of organic supporting structures includepolysaccharides, polymers or oligomers of organic synthetic polymers(such as, polyvinyl alcohol or polylactide). It is preferred that thesupporting structure possess substantially the same size and dimensionsas the peptide backbone or supporting structure. This can be determinedby calculating or measuring the size of the atoms and bonds of thepeptide and peptidomimetic. In one embodiment, the nitrogen of thepeptide bond can be substituted with oxygen or sulfur, for example,forming a polyester backbone. In another embodiment, the carbonyl can besubstituted with a sulfonyl group or sulfinyl group, thereby forming apolyamide (e.g., a polysulfonamide). Reverse amides of the peptide canbe made (e.g., substituting one or more-CONH-groups for a-NHCO-group).In yet another embodiment, the peptide backbone can be substituted witha polysilane backbone.

These compounds can be manufactured by known methods. For example, apolyester peptidomimetic can be prepared by substituting a hydroxylgroup for the corresponding α-amino group on amino acids, therebypreparing a hydroxyacid and sequentially esterifying the hydroxyacids,optionally blocking the basic and acidic side chains to minimize sidereactions. Determining an appropriate chemical synthesis route cangenerally be readily identified upon determining the chemical structure.

Peptidomimetics can be synthesized and assembled into librariescomprising a few to many discrete molecular species. Such libraries canbe prepared using well-known methods of combinatorial chemistry, and canbe screened to determine if the library comprises one or morepeptidomimetics which have the desired activity. Such peptidomimeticantagonists can then be isolated by suitable methods.

Nucleic Acid Antagonists

PLVAP antagonists also include various nucleic acids, including nucleicacid molecules that inhibit PLVAP gene expression (e.g., siRNA,antisense oligonucleotides, ribozymes). For example, small interferingribonucleic acids (siRNAs) and, similarly, short hairpin ribonucleicacids (shRNAs), which are processed into short siRNA-like molecules in acell, can prevent the expression (translation) of the PLVAP protein.siRNA molecules can be polynucleotides that are generally about 20 toabout 25 nucleotides long and are designed to bind a specific RNAsequence (e.g., a PLVAP mRNA sequence). siRNAs silence gene expressionin a sequence-specific manner, binding to a target RNA (e.g., an RNAhaving the complementary sequence) and causing the RNA to be degraded byendoribonucleases. siRNA molecules able to inhibit the expression of thePLVAP gene product can be produced by suitable methods. There areseveral algorithms that can be used to design siRNA molecules that bindthe sequence of a gene of interest (see, e.g., Mateeva O. et al. NucleicAcids Res. 35(8):Epub, 2007; Huesken D. et al., Nat. Biotechnol.23:995-1001; Jagla B. et al., RNA 11:864-872, 2005; Shabalinea S. A. BMCBioinformatics 7:65, 2005; Vert J. P. et al. BMC Bioinformatics 7:520,2006). Expression vectors that can stably express siRNA or shRNA areavailable. (See, e.g., Brummelkamp, T. R., Science 296: 550-553, 2002,Lee, N S, et al., Nature Biotechnol. 20:500-505, 2002; Miyagishi, M.,and Taira, K. Nature Biotechnol. 20:497-500, 2002; Paddison, P. J., etal., Genes & Dev. 16:948-958, 2002; Paul, C. P., et al., NatureBiotechnol. 20:505-508; 2002; Sui, G., et al., Proc. Natl. Acad. Sci.USA 99(6):5515-5520, 2002; Yu, J-Y, et al., Proc. Natl. Acad. Sci. USA99(9):6047-6052, 2002; Elbashir, S M, et al., Nature 411:494-498,2001.). Stable expression of siRNA/shRNA molecules is advantageous inthe treatment of cancer as it enables long-term expression of themolecules, potentially reducing and/or eliminating the need for repeatedtreatments.

Antisense oligonucleotides (e.g., DNA, riboprobes) can also be used asPLVAP antagonists to inhibit PLVAP expression. Antisenseoligonucleotides are generally short (˜13 to ˜25 nucleotides)single-stranded nucleic acids which specifically hybridize to a targetnucleic acid sequence (e.g., mRNA) and induce the degradation of thetarget nucleic acid (e.g., degradation of the RNA through RNaseH-dependent mechanisms) or sterically hinder the progression of splicingor translational machinery. (See, e.g., Dias N. and Stein C. A., Mol.Can. Ther. 1:347-355, 2002). There are a number of different types ofantisense oligonucleotides that can be used as PLVAP antagonistsincluding methylphosphonate oligonucleotides, phosphorothioateoligonucleotides, oligonucleotides having a hydrogen at the 2′-positionof ribose replaced by an O-alkyl group (e.g., a methyl), polyamidenucleic acid (PNA), phosphorodiamidate morpholino oligomers (deoxyribosemoiety is replaced by a morpholine ring), PN (N3′→P5′ replacement of theoxygen at the 3′ position on ribose by an amine group) and chimericoligonucleotides (e.g., 2′-O-Methyl/phosphorothioate). Antisenseoligonucleotides can be designed to be specific for a protein usingpredictive algorithms. (See, e.g., Ding, Y., and Lawrence, C. E.,Nucleic Acids Res., 29:1034-1046, 2001; Sczakiel, G., Front. Biosci.,5:D194-D201, 2000; Scherr, M., et al., Nucleic Acids Res., 28:2455-2461,2000; Patzel, V., et al. Nucleic Acids Res., 27:4328-4334, 1999; Chiang,M. Y., et al., J. Biol. Chem., 266:18162-18171, 1991; Stull, R. A., etal., Nucleic Acids Res., 20:3501-3508, 1992; Ding, Y., and Lawrence, C.E., Comput. Chem., 23:387-400, 1999; Lloyd, B. H., et al., Nucleic AcidsRes., 29:3664-3673, 2001; Mir, K. U., and Southern, E. M., Nat.Biotechnol., 17:788-792, 1999; Sohail, M., et al., Nucleic Acids Res.,29:2041-2051, 2001; Altman, R. K., et al., J. Comb. Chem., 1:493-508,1999). The antisense oligonucleotides can be produced by suitablemethods; for example, nucleic acid (e.g., DNA, RNA, PNA) synthesis usingan automated nucleic acid synthesizer (from, e.g., Applied Biosystems)(see also Martin, P., Helv. Chim. Acta 78:486-504, 1995). Antisenseoligonucleotides can also be stably expressed in a cell containing anappropriate expression vector.

Antisense oligonucleotides can be taken up by target cells (e.g., tumorcells) via the process of adsorptive endocytosis. Thus, in the treatmentof a subject (e.g., mammalian), antisense PLVAP oligonucleotides can bedelivered to target cells (e.g., tumor cells) by, for example, injectionor infusion. For instance, purified oligonucleotides or siRNA/shRNA canbe administered alone or in a formulation with a suitable drug deliveryvehicle (e.g., liposomes, cationic polymers, (e.g., poly-L-lysine, PAMAMdendrimers, polyalkylcyanoacrylate nanoparticles and polyethyleneimine))or coupled to a suitable carrier peptide (e.g., homeotic transcriptionfactor, the Antennapedia peptide, Tat protein of HIV-1, E5CA peptide).

Ribozymes can also be used as PLVAP antagonists to inhibit PLVAPexpression. Ribozymes are RNA molecules possessing enzymatic activity.One class of ribozymes is capable of repeatedly cleaving other separateRNA molecules into two or more pieces in a nucleotide base sequencespecific manner. See Kim et al., Proc Natl Acad Sci USA, 84:8788 (1987);Haseloff & Gerlach, Nature, 334:585 (1988); and Jefferies et al.,Nucleic Acid Res, 17:1371 (1989). Such ribozymes typically have twofunctional domains: a catalytic domain and a binding sequence thatguides the binding of ribozymes to a target RNA through complementarybase-pairing. Once a specifically-designed ribozyme is bound to a targetmRNA, it enzymatically cleaves the target mRNA, typically reducing itsstability and destroying its ability to directly translate an encodedprotein. After a ribozyme has cleaved its RNA target, it is releasedfrom that target RNA and thereafter can bind and cleave another target.That is, a single ribozyme molecule can repeatedly bind and cleave newtargets.

In accordance with the present invention, a ribozyme may target anyportion of the mRNA encoding PLVAP. Methods for selecting a ribozymetarget sequence and designing and making ribozymes are generally knownin the art. See, e.g., U.S. Pat. Nos. 4,987,071; 5,496,698; 5,525,468;5,631,359; 5,646,020; 5,672,511; and 6,140,491, each of which isincorporated herein by reference in its entirety. For example, suitableribozymes may be designed in various configurations such as hammerheadmotifs, hairpin motifs, hepatitis delta virus motifs, group I intronmotifs, or RNase P RNA motifs. See, e.g., U.S. Pat. Nos. 4,987,071;5,496,698; 5,525,468; 5,631,359; 5,646,020; 5,672,511; and U.S. Pat. No.6,140,491; Rossi et al., AIDS Res Human Retroviruses 8:183 (1992);Hampel & Tritz, Biochemistry 28:4929 (1989); Hampel et al., NucleicAcids Res, 18:299 (1990); Perrotta & Been, Biochemistry 31:16 (1992);and Guerrier-Takada et al., Cell, 35:849 (1983).

Ribozymes can be synthesized by the same methods used for normal RNAsynthesis. For example, suitable methods are disclosed in Usman et al.,J Am Chem Soc, 109:7845-7854 (1987) and Scaringe et al., Nucleic AcidsRes, 18:5433-5441 (1990). Modified ribozymes may be synthesized by themethods disclosed in, e.g., U.S. Pat. No. 5,652,094; InternationalPublication Nos. WO 91/03162; WO 92/07065 and WO 93/15187; EuropeanPatent Application No. 92110298.4; Perrault et al., Nature, 344:565(1990); Pieken et al., Science, 253:314 (1991); and Usman & Cedergren,Trends Biochem Sci, 17:334 (1992).

PLVAP antagonists of the invention can also be nucleic acid molecules(e.g., oligonucleotides) that bind to, and inhibit the activity of, aPLVAP protein. Suitable nucleic acid PLVAP antagonists include aptamers,which are capable of binding to a particular molecule of interest (e.g.,human PLVAP) with high affinity and specificity through interactionsother than classic Watson-Crick base pairing (Tuerk and Gold, Science249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).

Aptamers, like peptides generated by phage display or monoclonalantibodies (MAbs), are capable of specifically binding to selectedtargets and, through binding, block their targets' ability to function.Created by an in vitro selection process from pools of random sequenceoligonucleotides, aptamers have been generated for over 100 proteinsincluding growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(30-45 nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates against closely related targets (e.g., will typically notbind other proteins from the same gene family). A series of structuralstudies have shown that aptamers are capable of using the same types ofbinding interactions (hydrogen bonding, electrostatic complementarity,hydrophobic contacts, steric exclusion, etc.) that drive affinity andspecificity in antibody-antigen complexes.

An aptamer that binds to a target of interest (e.g., a human PLVAPprotein) can be generated and identified using a standard process knownas “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX),described in, e.g., U.S. Pat. Nos. 5,475,096 and 5,270,163.

Identification of PLVAP Antagonists

Agents having binding specificity for PLVAP gene products can beidentified in a screen, for example, a high-throughput screen ofchemical compounds and/or libraries (e.g., chemical, peptide, nucleicacid libraries).

Antibodies that specifically bind human PLVAP can be identified, forexample, by screening commercially available combinatorial antibodylibraries (Dyax Corp., MorphoSys AG). Suitable combinatorial antibodylibraries and standard methods of screening these libraries aredescribed in Hoet et al., Nature Biotechnology 23(3):344-348 (2005) andRauchenberger et al., J. Biol. Chem. 278(40):38194-38205 (2003), thecontents of which are incorporated herein by reference. Such librariesor collections of molecules can also be prepared using well-knownchemical methods.

Alternatively murine antibodies that specifically bind human PLVAP canbe identified, for example, by immunizing mice with PLVAP proteins,protein fragments or peptides, along with an adjuvant to break toleranceto the antigen. These antibodies can be screened for the desiredspecificity and activity and then humanized using known techniques tocreate suitable agents for the treatment of human disease.

Compounds or small molecules can be identified from numerous availablelibraries of chemical compounds from, for example, the ChemicalRepository of the National Cancer Institute and the Molecular LibrariesSmall Molecules Repository (PubChem), as well as libraries of theInstitute of Chemistry and Cell Biology at Harvard University and otherlibraries that are available from commercial sources (e.g., Chembridge,Peakdale, CEREP, MayBridge, Bionet). Such libraries or collections ofmolecules can also be prepared using well-known chemical methods, suchas well-known methods of combinatorial chemistry. The libraries can bescreened to identify compounds that bind and inhibit PLVAP.

Identified compounds can serve as lead compounds for furtherdiversification using well-known methods of medicinal chemistry. Forexample, a collection of compounds that are structural variants of thelead can be prepared and screened for PLVAP binding and/or inhibitoryactivity. This can result in the development of a structure activityrelationship that links the structure of the compounds to biologicalactivity. Compounds that have suitable binding and inhibitory activitycan be developed further for in vivo use.

Agents that bind PLVAP can be evaluated further for PLVAP antagonistactivity. For example, a composition comprising a PLVAP protein can beused in a screen or binding assay to detect and/or identify agents thatbind and antagonize the PLVAP protein. Compositions suitable for useinclude, for example, cells that naturally express a PLVAP protein(e.g., liver vascular endothelial cells), extracts of such cells, andrecombinant PLVAP protein.

An agent that binds a PLVAP protein can be identified in a competitivebinding assay, for example, in which the ability of a test agent toinhibit the binding of PLVAP to a reference agent is assessed. Thereference agent can be a full-length PLVAP protein or a portion thereof.The reference agent can be labeled with a suitable label (e.g.,radioisotope, epitope label, affinity label (e.g., biotin and avidin orstreptavadin), spin label, enzyme, fluorescent group, chemiluminescentgroup, dye, metal (e.g., gold, silver), magnetic bead) and the amount oflabeled reference agent required to saturate the PLVAP protein in theassay can be determined. The specificity of the formation of the complexbetween the PLVAP protein and the test agent can be determined using asuitable control (e.g., unlabeled agent, label alone).

The capacity of a test agent to inhibit formation of a complex betweenthe reference agent and a PLVAP protein can be determined as theconcentration of test agent required for 50% inhibition (IC₅₀ value) ofspecific binding of labeled reference agent. Specific binding ispreferably defined as the total binding (e.g., total label in complex)minus the non-specific binding. Non-specific binding is preferablydefined as the amount of label still detected in complexes formed in thepresence of excess unlabeled reference agent. Reference agents suitablefor use in the method include molecules and compounds which specificallybind to PLVAP, e.g., an antibody that binds PLVAP.

An agent that antagonizes a PLVAP protein can be identified by screeningfor agents that have an ability to antagonize (reduce, prevent, inhibit)one or more activities of PLVAP, such as, for example, tumorvascularization. Such activities can be assessed by one of skill in theart using any appropriate in vitro or in vivo assay.

Pharmaceutical Compositions

A PLVAP antagonist of the invention can be administered to a mammaliansubject as part of a pharmaceutical or physiological composition, forexample, as part of a pharmaceutical composition comprising a PLVAPantagonist and a pharmaceutically acceptable carrier. Formulations orcompositions comprising a PLVAP antagonist (e.g., an antibody thatspecifically binds PLVAP) or compositions comprising a PLVAP antagonistand one or more other therapeutic agents (e.g., a chemotherapeuticagent, for example, doxorubicin, 5-fluorouracil, tamoxifen, octreotide)will vary according to the route of administration selected (e.g.,solution, emulsion or capsule). Suitable pharmaceutical carriers cancontain inert ingredients which do not interact with the PLVAPantagonist. Standard pharmaceutical formulation techniques can beemployed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceuticalcarriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containingabout 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank'ssolution, Ringer's lactate and the like. Formulations can also includesmall amounts of substances that enhance the effectiveness of the activeingredient (e.g., emulsifying, solubilizing, pH buffering, wettingagents). Methods of encapsulation compositions (such as in a coating ofhard gelatin or cyclodextran) are known in the art. For inhalation, theagent can be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer or nebulizer or pressurized aerosoldispenser).

Diagnostic Kits

The invention also provides diagnostic kits for detecting the presenceof a hepatocellular carcinoma in a subject. Such kits comprise at leastone agent (e.g., a nucleic acid probe, an antibody) for detecting PLVAPgene expression in a sample (e.g., a biological sample from a mammaliansubject). PLVAP gene expression can be detected, for example, bydetecting a PLVAP gene product, such as a PLVAP mRNA or a PLVAP protein,in the sample.

Accordingly, in one embodiment, the kit comprises at least one nucleicacid probe (e.g., an oligonucleotide probe) that specifically hybridizesto a PLVAP RNA (e.g., mRNA, hnRNA) transcript. Such probes are capableof hybridizing to PLVAP RNA under conditions of high stringency.

In another embodiment, the kit includes a pair of oligonucleotideprimers that are capable of specifically hybridizing to a PLVAP geneproduct (e.g., mRNA, cDNA) in a sample. Such primers can be used in anystandard nucleic acid amplification procedure (e.g., polymerase chainreaction (PCR), for example, RT-PCR, quantitative real time PCR) todetermine the level of the PLVAP gene product in the sample.

In another embodiment, the kits of the invention include an antibodythat specifically binds a PLVAP protein (e.g., a human PLVAP protein).Such antibodies include any of the PLVAP antibodies of the inventiondescribed herein. In one embodiment, the antibody comprises a V_(H)domain having the amino acid sequence of SEQ ID NO:4 and a V_(L) domainhaving the amino acid sequence of SEQ ID NO:9. In another embodiment,the antibody comprises a V_(H) domain having the amino acid sequence ofSEQ ID NO:14 and a V_(L) domain having the amino acid sequence of SEQ IDNO:19.

The diagnostic agents in the kits of the invention can include one ormore labels (e.g., detectable labels). Numerous suitable labels fordiagnostic agents are known in the art and include, but are not limitedto, any of the labels described herein. In a particular embodiment, thediagnostic agent (e.g., antibody) includes a radioisotope, such thatagent can be used for immuno-positron emission tomography (immuno-PET).

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

EXEMPLIFICATION Example 1 PLVAP Expression is Elevated in HCC LiverTissues Relative to Non-HCC Liver Tissues

Materials and Methods:

Tissue Samples

Tissues of HCC and adjacent non-tumorous liver were collected from freshspecimens surgically removed from human patients for therapeuticpurpose. These specimens were collected under direct supervision ofattending pathologists. The collected tissues were immediately stored inliquid nitrogen at the Tumor Bank of the Koo Foundation Sun Yat-SenCancer Center (KF-SYSCC). Paired tissue samples from eighteen HCCpatients were available for the study. The study was approved by theInstitutional Review Board and written informed consent was obtainedfrom all patients. The clinical characteristics of the eighteen HCCpatients from this study are summarized in Table 1.

TABLE 1 Clinical data for eighteen HCC patients from which paired HCCand adjacent non-tumorous liver tissue samples were obtained Case HCVTNM AFP No Sex Age HBsAg HBsAb IgG Stage (ng/ml) Differentiation 1 M70 + − 2 2 Moderate 2 M 75 − + + 4A 5 Well 3 M 59 + − 4A 1232 Moderate 4F 53 + + 1 261 Moderate 5 M 45 + − 2 103 Moderate 6 M 57 + + − 2 5Moderate 7 M 53 + + − 3A 19647 Moderate 8 M 54 − − + 3A 7 Moderate 9 M44 + − 4A 306 Moderate 10 M 76 − − + 3A 371 Moderate 11 F 62 + − − 3A302 Moderate 12 F 73 − − + 2 42 Moderate 13 M 46 + − 4A 563 Moderate 14M 45 − − 3A 64435 Moderate 15 M 41 + − 2 33.9 Well 16 M 44 + + − 2 350Moderate 17 M 67 + − 3A 51073 Moderate 18 M 34 + − 4A 2331 ModeratemRNA Transcript Profiling

Total RNA was isolated from tissues frozen in liquid nitrogen usingTrizol® reagents (Invitrogen, Carlsbad, Calif.). The isolated RNA wasfurther purified using RNAEasy® Mini kit (Qiagen, Valencia, Calif.) andits quality assessed using the RNA 6000 Nano assay in an Agilent 2100Bioanalyzer (Agilent Technologies, Waldbronn, Germany). All RNA samplesused for the study had an RNA Integrity Number (RIN) greater than 5.7(8.2±1.0, mean±SD). Hybridization targets were prepared from 8 μg totalRNA according to Affymetrix® protocols and hybridized to an Affymetrix®U133A GeneChip®, which contains 22,238 probe-sets for approximately13,000 human genes. Immediately following hybridization, the hybridizedarray underwent automated washing and staining using an Affymetrix®GeneChip® fluidics station 400 and the EukGE WS2v4 protocol. Thereafter,U133A GeneChip® microarray chips were scanned in an Affymetrix®GeneArray scanner 2500.

Determination of Present and Absent Call of Microarray Data

Affymetrix® Microarray Analysis Suite (MAS) 5.0 software was used togenerate present calls for the microarray data for all 18 pairs of HCCand adjacent non-tumor liver tissues. All parameters for present calldetermination were default values. Each probe-set was determined as“present,” “absent” or “marginal” by MAS 5.0. Similarly, the samemicroarray data were processed using dChip version-2004 software todetermine “present,” “absent” or “marginal” status for each probe-set onthe microarrays.

Identification of Probe-Sets with Extreme Differential Expression

For identification of genes with extreme differential expression betweenHCC and adjacent non-tumor liver tissue, software written usingPractical Extraction and Report Language (PERL) was used according tothe following rules: “Tumor-specific genes” were defined as probe-setsthat were called “present” in HCC and “absent” or “marginal” in theadjacent non-tumor liver tissue by both MAS 5.0 and dChip. “Non-tumorliver tissue-specific genes” were defined as probe-sets called ‘absent’or ‘marginal’ in HCC and ‘present’ in the paired adjacent non-tumorliver tissue by both MAS 5.0 and dChip. A flowchart diagram depictingthe identification algorithm is shown in FIG. 1.

Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction(RT-PCR)

TaqMan™ real-time quantitative reverse transcriptase-PCR (qRT-PCR) wasused to quantify mRNA. cDNA was synthesized from 8 μg of total RNA foreach sample using 1500 ng oligo(dT) primer and 600 units SuperScript™ IIReverse Transcriptase from Invitrogen (Carlsbad, Calif.) in a finalvolume of 60 μl according to the manufacturer's instructions. For eachRT-PCR reaction, 0.5 μl cDNA was used as template in a final volume of25 μl following the manufacturers' instructions (ABI and Roche). The PCRreactions were carried out using an Applied Biosystems 7900HT Real-TimePCR system. Probes and reagents required for the experiments wereobtained from Applied Biosystems (ABI) (Foster City, Calif.). Thesequences of primers and the probes used for real-time quantitativeRT-PCR of PLVAP are 5′-CCTGCAGGCATCCCTGTA-3′ (forward primer) (SEQ IDNO:25); 5′-CGGGCCATCCCTTGGT-3′ (reverse primer) (SEQ ID NO:26); and5′-CCCCATCCAGTGGCTG-3′ (probe) (SEQ ID NO:27). Hypoxanthine-guaninephosphoribosyltransferase (HPRT) housekeeping gene was used as anendogenous reference for normalization. All samples were run induplicate on the same PCR plate for the same target mRNA and theendogenous reference HPRT mRNA. The relative quantities of target mRNAswere calculated by comparative Ct method according to manufacturer'sinstructions (User Bulletin #2, ABI Prism® 7700 Sequence DetectionSystem). A non-tumorous liver sample was chosen as the relativecalibrator for calculation.

Results:

The PLVAP gene expression intensities in 18 pairs of HCC and adjacentnon-tumorous liver tissues are shown in FIG. 2. The average geneexpression intensities were 759.8±436.5 and 170.6±53.4 (mean±SD) forpaired HCC and adjacent non-tumorous liver tissue, respectively. The pvalue of paired t-test between the two groups was 2.8×10⁻⁵. Theseresults indicate that PLVAP is expressed in HCC and not in non-tumorousliver tissue. This elevated expression of PLVAP in HCC was furtherconfirmed when 82 unpaired HCC samples showed an average expressionintensity of 810.4±482.0 (mean±SD), which is essentially the same as thefinding from the 18 paired HCC samples (p=0.62 by t-test) (FIG. 2).

In order to confirm that PLVAP is significantly expressed in HCC livertissue and not in non-tumorous liver tissue, real-time quantitativeRT-PCR was performed on RNA samples from 18 pairs of HCC and adjacentnon-tumorous liver tissue. Quantities of PLVAP mRNA were significantlyhigher in HCC relative to non-tumorous liver tissues (see FIG. 3A andTable 2). Although the results showed some overlap between two groups,PLVAP transcripts were higher in HCC than in adjacent non-tumorous livertissue within the same individual for all individuals tested except one(FIG. 3B). This exception was likely associated with uneven degrees ofRNA degradation during storage process of tissues.

TABLE 2 PLVAP gene expression intensities for 18 pairs of HCC andadjacent non-tumorous liver tissue Expression Intensity* Sample Adjacentnon-tumorous Number HCC liver tissue 1 1757 195 2 1329 210 3 1148 168 41130 211 5 1096 213 6 1068 181 7 932 101 8 804 60 9 630 155 10 612 17511 607 125 12 519 146 13 478 300 14 422 180 15 275 105 16 251 204 17 251155 18 186 184

Example 2 PLVAP is Specifically Expressed by HCC Vascular EndothelialCells

Materials and Methods:

Laser Capture Microdissection (LCM) of Formalin-Fixed Paraffin EmbeddedTissues

LCM of formalin fixed tissue from paraffin blocks was carried out usingArcturus PixCell® IIe system, CapSure™ HS LCM caps, and Paradise™reagent system from Arcturus Bioscience, Inc. (Mountain View, Calif.).Seven micrometer thick tissue sections were cut, deparaffinized,rehydrated, stained and dehydrated for LCM according to manufacturer'sinstructions. Target cells were captured onto CapSure™ HS LCM caps using7.5 μm laser spot size at 50 mW power and 1.3 ms duration. Approximately5000 to 6000 cells were captured on each cap. However, only 1000 to 2000hepatocellular carcinoma vascular endothelial cells were captured ontoeach cap due to paucity of cells.

RNA Extraction from LCM Tissue Sections for Quantitative RT-PCR

Cells captured onto the CapSure™ HS LCM caps as described above wereprocessed for RNA extraction, cDNA synthesis, in vitro transcription andantisense RNA amplification using the Paradise™ reagent system accordingto manufacturer's instructions. The synthesized anti-sense RNA was thenused as a template for two-step TaqMan® real time quantitative RT-PCRfor quantitation of PLVAP and beta-actin mRNA in the cells captured byLCM. The first step (i.e., reverse transcription) was carried out using4.5 μl anti-sense RNA and TaqMan® Reverse Transcription Reagents (ABI)in a final volume of 10 μl following the manufacturer's protocol. Thesecond step (i.e., real-time PCR) was performed using 2.4 μl of cDNAtemplate, the primers/probe mix and the TaqMan® universal PCR Master Mixfrom Applied Biosystems in a final volume of 25 μl. Real-time PCR wascarried out in a Smart Cycler® II machine (Cephid, Inc., Sunnyvale,Calif.). The reactions were initially incubated at 50° C. for 2 minutesand then at 95° C. for 10 minutes. Thereafter, 45 cycles of denaturationat 95° C. for 15 seconds and annealing/extension at 60° C. for 40seconds were performed. The sequences of the primers and the probes arelisted in Table 3.

TABLE 3 Primer and probe sequences for real-time quantitative RT-PCR forPLVAP and beta-actin levels in samples prepared by laser-capturedmicrodissection PLVAP gene beta-Actin gene forward5′-CCTTGAGCGTGAGTGTTTCCA-3′ 5′-GTCCCCCAACTTGAGATGTATGAAG-3′ primer(SEQ ID NO: 28) (SEQ ID NO: 29) reverse 5′-GGCAGGGCTGGGAGTTG-3′5′-GTCTCAAGTCAGTGTACAGGTAAGC-3′ primer (SEQ ID NO: 30) (SEQ ID NO: 31)Taqman 5′-CTCCCAGGGAGACCAA-3′ 5′-AAGGAGTGGCTCCCCTCC-3′ probe(SEQ ID NO: 32) (SEQ ID NO: 33)Preparation of Expression Vector for Recombinant Fusion PLVAP₅₁₋₄₄₂Protein

Plasmid pGEM®-T Easy-PLVAP₅₁₋₄₄₂ was generated by inserting a PCRfragment encoding amino acid residues 51 to 442 of PLVAP into thepGEM®-T Easy Vector (Promega, Inc., Madison, Wis.). The PCR fragment wasamplified from a cDNA clone of PLVAP from OriGene (Rockville, Md.) byusing the primer set of 5′-

AACGTGCACGTGAGCACAGAGTCC-3′ (SEQ ID NO:34) and 5′-

TGAGCATATCCCTGCATCCTCC-3′ (SEQ ID NO:35). For construction of plasmidpET-15b-PLVAP₅₁₋₄₄₂, a cDNA fragment encoding amino acid residues 51 to442 of PLVAP with NdeI and BamHI recognition sequences at eachrespective end was excised from pGEM®-T Easy-PLVAP₅₁₋₄₄₂ and insertedinto pET-15b (Novagen, Inc., San Diego, Calif.). The expressionconstruct described above was verified by DNA sequencing.

Expression and Purification of Recombinant Fusion PLVAP₅₁₋₄₄₂ Protein

For production of recombinant His-tagged PLVAP₅₁₋₄₄₂ protein (SEQ IDNO:2) (FIG. 4), Escherichia coli (Rosetta-Gami™2(DE3)pLysS cells)(Novagen) was transformed by incubating competent cells withpET-15b-PLVAP₅₁₋₄₄₂ plasmid DNA on ice for 5 min, followed by incubationin a 42° C. water bath for 30 s and then again on ice for 2 min. Priorto plating on selective medium, the transformants were incubated at 37°C. while shaking at 250 rpm with SOC medium (0.5% Yeast Extract; 2%Tryptone; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl₂; 10 mM MgSO₄; 20 mMGlucose) for 60 min. Expression of His-tagged fusion protein inRosetta-Gami™2(DE3)pLysS Escherichia coli was induced with 1 mMisopropyl-B-D-thiogalactopyranoside for 16 hours at 30° C. Following theinduction, the bacterial cells were subjected to lysis by sonication inequilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7)supplemented with 8 M urea and separated into soluble and insolublefractions by centrifugation at 5,600×g for 30 minutes. For furtherpurification of the His-PLVAP₅₁₋₄₄₂ protein, soluble fraction was loadedon a TALON® Metal Affinity Resin (Clontech, Inc., Palo Alto, Calif.),washed with equilibration buffer and eluted with elution buffer (50 mMsodium phosphate, 300 mM NaCl, pH 7, 250 mM imidazole). The His-tag ofthe purified fusion protein was removed by thrombin cleavage (Novagen)according to manufacturer's instructions (see FIG. 5). The resultingPLVAP₅₁₋₄₄₂ protein was recovered by extensive dialysis against PBS. Toverify the identity of the recombinant PLVAP protein, a small quantityof mouse antiserum against GST-PLVAP₃₃₁₋₄₃₀ fusion protein was purchasedfrom the Biodesign Insitute (Tempe, Ariz.). The recombinant PLVAP₅₁₋₄₄₂protein without the His-tag was detected by Western blot analysis usingthis antibody, but did not react with antibodies to the His-tag. Theseresults confirm the identity of the recombinant PLVAP protein.

Generation of Mouse Anti-Human PLVAP Serum

Purified PLVAP₅₁₋₄₄₂ recombinant protein in PBS was used to immunize 6weeks old Balb/cByj mice. Each mouse was initially immunized withsubcutaneous injection at multiple sites with a total of 14 μgPLVAP₅₁₋₄₄₂ protein in complete Freund's adjuvant (Sigma, Inc., StLouis, Mo.). Thereafter, immunization was boosted with 7 μg PLVAP₅₁₋₄₄₂recombinant protein in incomplete Freund's adjuvant once every two weeksfor three times. A week after the last boosting immunization, mice werebled for preparation of antiserum.

Enzyme-Linked Immunosorbent Assay (ELISA)

Reagents and Solutions:

-   -   1. Recombinant PLVAP protein    -   2. Anti-mouse IgG-alkaline phosphatase conjugate (Cat. #:        AP124A, CHEMICON)    -   3. Coating buffer (0.137 M Sodium Chloride, 0.01 M Sodium        Phosphate Dibasic Heptahydrate, 2 mM Potassium Phosphate        Monobasic, 0.002% (0.3 mM) Sodium azide, pH 7.2-7.4)    -   4. Washing buffer (0.137 M Sodium Chloride, 0.01 M Sodium        Phosphate Dibasic Heptahydrate, 2 mM Potassium Phosphate        Monobasic, 0.2% Tween20 (Cat. #: P1379, SIGMA, pH 7.2-7.4)    -   5. Blocking buffer (0.137 M Sodium Chloride, 0.01 M Sodium        Phosphate Dibasic Heptahydrate, 2 mM Potassium Phosphate        Monobasic, 2% Bovine Serum Albumin (Cat. #: 82-045, PENTEX),        0.05% Tween20 (Cat. #: P1379, SIGMA), pH 7.2-7.4)    -   6. Carbonate buffer (0.016 M Sodium Bicarbonate, 0.014 M Sodium        Carbonate, 2 mM Magnesium Chloride, 0.002% (0.3 mM) Sodium        Azide, pH 9.6)    -   7. Akaline Phosphatase substrate: One 40 mg phosphatase        substrate tablet (Cat. #: P5994, SIGMA) dissolved in 40 ml        carbonate buffer        Procedure:

The titers of antibodies in the anti-PLVAP sera were determined usingELISA. First, the 96 well ELISA plate was coated with 50 μl of PLVAPprotein dissolved in Phosphate buffered saline (PBS) containing 0.002%sodium azide (i.e., coating buffer) at a concentration in the range of2.5 μg/m overnight at 4° C. After three washes with 200 μl of washingbuffer (PBS containing 0.05% Tween-20), each well of the coated platewas blocked with 150 μl blocking buffer (i.e., washing buffer containing2% bovine serum albumin) at room temperature for 30 minutes. After threefurther washes, each well was incubated with 50 μl of diluted antiserum(serial two fold dilution from 1,000× to 128,000×) prepared in thedilution buffer for 45 minutes at room temperature. Thereafter, eachwell was incubated with anti-mouse IgG alkaline phosphatase conjugate at5,000× dilution (Chemico, Inc., Temecula, Calif.) for 30 minutes at roomtemperature. After three washes, the bound antibodies were quantifiedwith 100 μl alkaline phosphatase substrate (Sigma, Inc., St Louis, Mo.)and measurement of absorbance was performed at 405 nm after anincubation period of 25 to 40 min. using an ELISA plate reader.

Immunohistochemical (IHC) Detection of PLVAP in Formalin-Fixed Tissues

Six micrometer sections were cut from paraffin blocks of formalin-fixedtissues. The sections were mounted on SuperFrost™ plus adhesion glassslides (Menzel Glaser GmbH, Braunschweig, Germany). The sections thenwere processed for immunostaining of PLVAP in a Benchmark XT automatedstaining instrument (Ventana Medical Systems, Inc., Tucson, Ariz.) usingXT-iView-DAB-V.1 protocol with mild CCI conditioning for 30 minutes andsections were incubated with 400× diluted anti-human PLVAP serum at 37°C. for 36 minutes. The second antibody and the reagents used to detectbinding of mouse anti-human PLVAP antibodies were from the iView™DABDetection Kit from Ventana Medical Systems, Inc. (Tucson, Ariz.). Allreagents and buffers were purchased from Ventana Medical Systems.

Results:

To determine the cellular source of PLVAP in HCC samples, HCC vascularendothelial cells, tumor cells of hepatocellular carcinoma andnon-tumorous hepatocytes, including lining sinusoidal endothelial cells,were dissected out of the samples using laser capture microdissection(LCM). Due to close apposition between hepatoma cells andcapillary-lining endothelial cells, effort was made to avoid inclusionof capillary-lining endothelial cells during dissection. The RNAsextracted from the dissected cells were used for two-step real timequantitative RT-PCR to determine the relative quantities of PLVAP mRNA.Specimens from two different patients were studied. The results shown inTable 4 and FIGS. 6A-6C indicate that PLVAP is expressed by HCC vascularendothelial cells (FIG. 6A), while no detectable PLVAP transcript wasdetected in adjacent non-tumorous liver tissues (FIG. 6B).

TABLE 4 Determination of PLVAP mRNA relative quantities in two HCCsamples by Taqman real time quantitative RT-PCR in cells dissected bylaser-capturing microdissection Relative Quantity of PLVAP mRNA AdjacentNon- HCC Endothelial tumorous Liver HCC Tumor HCC Sample Cells TissueCells A 1 0 0.002 B 1 0.001 0.057

In order to further investigate the tissue and disease specificity ofPLVAP expression, polyclonal antibodies for use in immunohistochemistry(IHC) studies were generated against the extracellular domain of humanPLVAP (amino acids 51 to 442). As shown in FIG. 7, antiserum obtainedfrom Balb/c mice that were immunized with recombinant PLVAP₅₁₋₄₄₂protein contained a high titer of anti-PLVAP antibodies.

The anti-PLVAP antiserum was then used to determine the localization ofPLVAP expression in tissue sections from patients with hepatocellularcarcinoma (n=7) (FIGS. 8A-8F and 9A-9F), focal nodular hyperplasia (n=4)(FIGS. 10A-10F), hepatic hemangioma (n=2) (FIGS. 11A and 11B), chronicactive hepatitis B (n=2) (FIGS. 12A and 12B) or C (n=4) (FIGS. 13A-13D),and metastatic cancer (n=4) (i.e., intrahepatic cholangiocarcinoma,metastatic colorectal adenocarcinoma, or metastatic ovarian carcinoma)(FIGS. 14A-14D). The results showed that only capillary endothelialcells of hepatocellular carcinomas expressed PLVAP protein (FIGS. 8A,8C, 8E, 9A, 9C, 9E and 9F). PLVAP protein was not expressed byendothelial cells lining the vascular sinusoids/capillary ofnon-tumorous liver tissues, including cirrhotic liver, liver of focalnodular hyperplasia (FIGS. 10A-10F), and chronic hepatitis (FIGS. 12Aand 12B; FIGS. 13A-13D). Endothelial lining cells of hepatic hemagiomadid not show significant expression of PLVAP, either (FIGS. 11A and11B). These results demonstrate that PLVAP is a vascular endothelialbiomarker that is specific for hepatocellular carcinoma, but not forother diseases of liver. Therefore, PLVAP can be used as a diagnosticmarker and therapeutic target for HCC.

Example 3 Production and Characterization of Mouse Monoclonal Antibodiesthat Specifically Bind PLVAP

Materials and Methods:

Immunization Procedures

Five six-week-old female Balb/cByJ mice were immunized initially with 20μg of purified recombinant PLVAP protein dissolved in 0.125 mL phosphatebuffered saline (PBS) and emulsified in an equal volume of completeFreund's adjuvant. The PLVAP-adjuvant mixture was injected in 0.05 mLvolumes into each of four separate subcutaneous sites on the ventralside of the mice near the axillary and inguinal lymphatics, as well as afifth subcutaneous site, which was located between the scapulae. Allmice received a booster immunization of 20 μg of recombinant PLVAPprotein injected intraperitoneally three times every two weeks. One weekafter the last booster immunization, test bleedings were taken tomeasure whether mice were producing sufficiently high titers ofanti-PLVAP antibodies (>10,000×). A solid-phase enzyme-linkedimmunosorbent assay (ELISA) was used for this purpose. The mouse thatproduced the highest titer of PLVAP antibody was selected for theproduction of hybridomas.

Development of Murine Monoclonal Anti-PLVAP Antibodies

Three days before the scheduled fusion experiment to produce hybridomas,the mouse that produced the highest titer of PLVAP antibody was injectedintravenously with 20 μg of recombinant PLVAP. Hybridomas producingmonoclonal antibodies (MAbs) against PLVAP were produced according to apreviously described protocol (see Unit 2.5 Production of MonoclonalAntibodies, in Current Protocols in Immunology, editors: Coligan J E,Kruisbeek A M, Margulies D H, Shevach E M, and Strober W. Published byJohn Wiley & Sons, Inc., New York, 2001) with minor modification.Specifically, spleen cells harvested from the immunized mouse were fusedwith SP2/0 myeloma cells at a ratio of 7.5:1 (spleen cell:myeloma cells)using 50% polyethylene glycol 1540. The fusion products were seeded into96-well flat-bottom tissue culture plates, andhypoxanthine-aminopterin-thymidine (HAT) selective medium was added thenext day. Seven to ten days later, the supernatants of growth-positivewells were screened for production of anti-PLVAP antibodies by ELISA.Hybridomas initially producing anti-PLVAP MAbs were expanded andre-screened. Hybridomas that showed continued production of antibodieswere cloned by the limiting dilution method. MAb isotypes weredetermined using an ELISA. Monoclonal antibodies were purified fromascites or culture media by Protein G affinity column chromatography(Unit 2.7 Purification and Fragmentation of Antibodies, in CurrentProtocols in Immunology, editors: Coligan J E, Kruisbeek A M, MarguliesD H, Shevach E M, and Strober W. Published by John Wiley & Sons, Inc.,New York, 2001).

ELISA Assay

ELISA assays were performed as described herein (see Example 2).

Determination of Binding Affinities

Binding affinities of KFCC-GY4 and KFCC-GY5 anti-PLVAP monoclonalantibodies were measured at the ANT Technology Co., Ltd. (Taipei,Taiwan) using ANTQ300 quartz crystal microbalance technology (Lin S., etal. J Immunol Methods 239:121-124 (2000)).

Isolation and Culture of Human Umbilical Vascular Endothelial Cells(HUVEC)

Isolation and culture of HUVEC were carried out according to theestablished protocol described in Baudin B, Brunee A, Bosselut N andVaubourdolle M. Nature Protocols 2:481-485 (2007). During themaintenance of endothelial cell culture, 1% gelatin (DIFCO, Corp.)dissolved in phosphate buffered saline was used to replace collagensolution for coating culture plates or coverslips.

Extraction of Hydrophobic Membrane Proteins of HUVEC by Triton X-114(TX-114) Containing Buffer

Five hundred thousand HUVEC were seeded in a 10 cm culture dish for 24hours. The cells were then stimulated with human VEGF at 40 ng/ml for anadditional 72 hours. The cultured cells were washed with 5 ml phosphatebuffered saline (PBS) twice. The cells then were detached and liftedfrom the dish by incubation with 1 ml PBS containing 2 mM EDTA, wereplaced into a centrifuge tube, and were collected by centrifugation at300×g for 5 minutes. There were approximately 2 million cells in thepellet produced by centrifugation. The cell pellets were re-suspended in200 μl ice cold 0.05 M Tris buffer containing 5 mM EDTA and 0.5% (v/v)Triton™ X-114 (TX-114) detergent, pH 7.4. The solubilized cellsuspension was incubated on ice with occasional gentle vortexing.Thereafter, the cells suspension was centrifuged at 10,000×g for 10minutes at 4° C. to remove insoluble cellular debris. The supernatantwas transferred to a clean microfuge tube and incubated at 37° C. for 5minutes. During the incubation, TX-114 became separated from the aqueousphase. The microfuge tube was then centrifuged at 1000×g for 10 minutesat room temperature, such that the TX-114 was centrifuged to the bottomof the tube. The aqueous phase at the top of the tube was removed andthe TX-114 pellet containing hydrophobic cellular proteins was dissolvedin 2×SDS acrylamide gel sample buffer in a final volume of 50 μl.Fifteen μl of sample was used for SDS acrylamide gel electrophoresis.

SDS Acrylamide Gel Electrophoresis, Preparation of Western Blot andImmunoblotting

The procedures are the same as previously described by Kao K J, ScornikJ C and McQueen C F. Human Immunol 27:285-297 (1990), with slightmodification. Detection of antibody binding on Western blots was carriedout using alkaline phosphatase chemiluminescent substrate and anLAS-4000 Luminescent Image Analyzer (Fujifilm Corp.).

Immunofluorescent Microscopy

Materials:

-   1) Primary Antibodies:    -   a) Normal mouse IgG (Sigma Corp., catalog #: I-5381) dissolved        in phosphate buffered saline (PBS) to 1 mg/mL as a stock        solution, diluted with PBS-0.5% BSA to a concentration of 5        μg/mL before use;    -   b) Monoclonal mouse anti-human von Willebrand factor (vWF)        (DakoCytomation Corp., catalog #: M0616) diluted 50× with PBS        containing 0.5% BSA before use;    -   c) Purified KFCC-GY4 and KFCC-GY5 anti-PLVAP monoclonal        antibodies were diluted to 5 μg/m with PBS containing 0.5% BSA        before use;-   2) Secondary antibody: FITC-conjugated Goat F(ab′)₂ anti-mouse IgG    (H&L) (Serotec Corp., catalog #: Star105F);-   3) VectaShield® Mounting Medium with DAPI (Vector Labs Corp.,    catalog #: H-1200);-   4) 100% Methanol (Merck Corp., catalog #: 1.06009); and-   5) Hank's Balanced Salt Solution (HBSS) (Gibco Corp., catalog #:    12065-056) diluted to 1× before use.    Procedure:

To prepare human umbilical cord vascular endothelial cells forimmunofluorescent study, fifty thousand cells were placed in each wellof a 24-well culture plate with a 1.5 cm sterile round coverslip placedat the bottom of each well. Each well contained 0.5 ml M199 culturemedia that was supplemented with 20% fetal calf serum, 1% L-glutamine,1% antibiotic/antimycotic solution, 50 μg/ml heparin and 75 μg/mendothelial cell growth supplement (Sigma Corp. E0760). Each coverslipwas pre-coated with 200 μl of 0.4 mg/ml calf skin collagen (Sigma Corp.C9791) in 0.04% acetic acid (v/v) overnight. The coverslips were thenwashed with sterile 1× phosphate buffered saline (PBS) and subsequentlyair-dried for use. Cells were cultured overnight and then stimulatedwith 40 ng/ml vascular endothelial growth factor (VEGF) for anadditional 72 hours. The cells on the coverslips were used for theimmunofluorescent procedure.

To stain the cells for immunofluorescent microscopy, the cells grown onthe coverslip in each well were washed with 0.5 ml 1×HBSS. The cellswere then fixed and permeabilized in 0.5 ml ice cold methanol for 5minutes. The fixed cells were washed 3 times with 0.5 ml 1×PBS for 5minutes per wash. The fixed cells were then blocked with 0.5 ml 1×PBScontaining 0.5% BSA for 1 hour at room temperature. The coverslipcontaining the fixed cells was removed and placed on top of 0.2 mldiluted primary antibody solution, which contained 5 μg/m normal IgG,KFCC-GY4 or KFCC-GY5 anti-PLVAP monoclonal antibody, or a 50× dilutionof anti-human vWF monoclonal antibody, with the fixed cells facing downand in contact with antibody solution. The antibody solution was placedon a piece of parafilm in a small covered plastic container. Thehumidity inside was maintained by placing a small piece of filter paperwetted with water.

After incubation at 37° C. for one hour in a humidified container, thecoverslip was removed and the cells on the coverslip were washed 3 timeswith 0.5 ml PBS for 5 minutes each time. The fixed cells were thenincubated with 0.2 ml 200×-diluted FITC-conjugated Goat F(ab′)₂anti-mouse IgG secondary antibody for 50 minutes at 37° C. as describedfor incubation with primary antibody solution. Thereafter, the cellswere washed 3 times with PBS as described above. The stained cells weremounted on a glass slide using VectaShield® anti-fade solution. Excessmounting media was removed from the edge of the coverslip and the edgewas sealed with nail polish. The stained cells were examined using afluorescent microscope.

Results:

Immunization of Balb/cByJ mice with recombinant human PLVAP protein ledto the development of hybridomas producing monoclonal antibodies (mAbs)that recognized human PLVAP protein. Two hybridomas were selected forfurther study. The antibodies produced by these hybridomas were namedKFCC-GY4 and KFCC-GY5. The sequences of the V_(H) and V_(L) domains ofmonoclonal antibodies KFCC-GY4 and KFCC-GY5, and the CDRs of thesedomains, are shown in FIGS. 15A and 15B and FIGS. 16A and 16B,respectively.

The hybridoma cell line referred to as KFCC-GY4 has the A.T.C.C. PatentDeposit Designation PTA-9963, having been deposited on Apr. 8, 2009. Thehybridoma cell line referred to as KFCC-GY5 has the A.T.C.C. PatentDeposit Designation PTA-9964, having been deposited on Apr. 8, 2009.

Both KFCC-GY4 and KFCC-GY5 monoclonal antibodies bound recombinant PLVAPprotein in ELISA (FIG. 17) and immunoblot (FIGS. 18C and 18D) assays.

These antibodies also specifically reacted with PLVAP protein inextracts from human umbilical cord vascular endothelial cells in animmunoblot assay (FIGS. 19B and 19D). In addition, immunofluorescencestaining experiments showed binding of KFCC-GY4 and KFCC-GY5 monoclonalantibodies to PLVAP-expressing human vascular endothelial cells (FIGS.20C and 20D).

Binding affinities (K_(d)) of the monoclonal antibodies for recombinantPLVAP protein were determined to be 0.41×10⁻⁷ M for KFCC-GY5 mAb and0.6×10⁻⁷ M for KFCC-GY4 mAb using ANTQ300 quartz crystal microbalance(Lin, et al. J. Immunol. Methods 239:121-124, 2000).

Immunohistochemistry experiments performed on hepatoma sections from theliver of two different hepatoma patients using KFCC-GY4 or KFCC-GY5monoclonal anti-PLVAP antibodies showed that the KFCC-GY5 monoclonalantibody produced a stronger signal in vascular endothelial cells (FIGS.21A and 21C) than the KFCC-GY4 monoclonal antibody (FIGS. 21B and 21D).

Immunohistochemistry experiments performed on adjacent hepatoma andnon-tumorous liver tissue sections from the liver of the same patientwere performed on samples from four different randomly selected hepatomapatients using the KFCC-GY4 monoclonal anti-PLVAP antibody. PLVAPexpression was detected in vascular endothelial cells of hepatomatissues (FIGS. 22A, 22C, 22E and 22G), but not adjacent non-tumorousliver tissues (FIGS. 22B, 22D, 22F and 22H).

Example 4 PLVAP Protein is Expressed on the Surfaces of VascularEndothelial Cells

Materials and Methods:

Immunofluorescent Microscopy

Reagents:

The reagents used for the following procedure are as described inExample 3, with the following modifications:

-   -   the 1×HBSS wash buffer contained 0.1% sodium azide, which was        used to prevent endocytosis of antibodies bound to the cell        surface; and    -   the KFCC-GY4 and KFCC-GY5 monoclonal anti-PLVAP antibodies were        diluted in the 1×HBSS wash buffer with 0.1% sodium azide.        Procedure:

Immunofluorescent staining of human umbilical cord vascular endothelialcells (HUVECs) was performed as described in Example 3, except that thecells were not fixed and permeabilized with methanol. Instead, afterincubation with anti-PLVAP monoclonal antibodies, the cells were washedand fixed with 4% paraformaldehyde at room temperature for 10 minutes.Following this incubation, the cells were washed 3 times, then wereincubated with FITC-conjugated Goat F(ab′)₂ anti-mouse IgG. After threeadditional washes, the cells were processed for immunofluorescentmicroscopy as described in Example 3.

Results:

Using the approach described above, only PLVAP protein expressed on thecell surface could be detected. The results of these experimentsrevealed that both KFCC-GY4 and KFCC-GY5 anti-PLVAP monoclonalantibodies bound to the surface of HCC vascular endothelial cells (FIGS.23B and 23C), indicating that PLVAP protein is expressed on the surfacesof these cells. These findings suggest that antibodies that specificallybind PLVAP with high affinity will be able to bind to the surface of HCCvascular endothelial cells upon injection into the blood vessels of ahepatocellular carcinoma tumor.

Example 5 Anti-Human PLVAP Monoclonal Antibodies Bind to PLVAP Proteinsin Non-Human Primate Species

Materials and Methods:

Tissue array slides were first prepared according to manufacturer'sinstructions by baking slides at 60° C. for 2 hours to remove sealingparaffin. Thereafter, the slides were processed for immunohistochemicalstaining as described in Example 2 (IHC Detection of PLVAP in FormalinFixed Tissues). The only modification was that the slides were incubatedwith 1 μg/ml KFCC-GY4 and -GY5 monoclonal antibodies for 48 minutes at37° C.

Results:

In order to determine whether non-human primates can be used to evaluatethe pharmacokinetics, pharmacodynamics and toxicity of the KFCC-GY4 andKFCC-GY5 mAbs, as well as other antibodies derived from these mAbs,immunohistochemistry staining of arrays of formalin-fixed normal tissuesfrom human, rhesus monkey and cynomolgus monkey were performed using theKFCC-GY4 and KFCC-GY5 antibodies (FIG. 26). The KFCC-GY4 and KFCC-GY5anti-human PLVAP monoclonal antibodies bound to both human and monkey(cynomolgus and rhesus) capillary endothelial cells in different tissueswith high degree of similarity (FIG. 26). Examples of KFCC-GY5 antibodybinding to human and monkey normal adrenal gland, kidney, brain andliver sections are shown in FIGS. 27A-27F2. Anti-human CD34 monoclonalantibody was also used as a positive control on human tissue sections tohighlight blood vessels. These results indicate that rhesus andcynomolgus monkeys are suitable for use in pre-clinical trial studies ofPLVAP antibodies.

Example 6 KFCC-GY4 and KFCC-GY5 Monoclonal Antibodies Bind to AntigenicEpitopes Between Amino Acids 282 and 482 in the C-Terminal Region ofHuman PLVAP Protein

Materials and Methods:

Molecular Cloning

Plasmids expressing His-tagged N-terminal (amino acids 51-292) orC-terminal (amino acids 282-442) portions of the extracellular domain ofhuman PLVAP (amino acids 51-442) were constructed as follows. pGEM®-TEasy-PLVAP₅₁₋₄₄₂ was treated with Eco RV and Stu I, self-ligated andgenerated the resulting plasmid pGEM®-T Easy-PLVAP₅₁₋₂₉₂. Forconstruction of plasmid pET-15b-PLVAP₅₁₋₂₉₂, a cDNA fragmentrepresenting the amino acid residues 51 to 292 of PLVAP with NdeI/BamHIrecognition sequences at the ends was excised from pGEM®-TEasy-PLVAP₅₁₋₂₉₂ and inserted into pET-15b (Novagen). To constructpET-15b-PLVAP₂₈₂₋₄₄₂, pET-15b-PLVAP₅₁₋₄₄₂ was treated with Nde I and SacI, followed by blunt-end ligation using T4 DNA polymerase. Theexpression constructs described above were verified by DNA sequencingand transformed into Escherichia coli (Rosetta-Gami™2(DE3)pLysS cells).

Recombinant Protein Expression and Immunoblotting

Expression of His-tagged fusion proteins in Escherichia coliRosetta-Gami™2(DE3)pLysS cells was induced with 1 mMisopropyl-B-D-thiogalactopyranoside for 16 hours at 30° C. Following theinduction, the bacterial cells were subjected to lysis by Laemmli samplebuffer. The resultant cell lysates were resolved by SDS-PAGE andsubjected to Coomassie blue staining and immunoblotting using standardprotocols and the following antibodies: primary antibodies: KFCC-GY4,KFCC-GY5, mAb 2A7-6, anti-his mAb (LTK Biotechnology, Taiwan).Immunoblots were developed by alkaline phosphatase-conjugated secondaryantibodies against mouse IgG from Chemicon, Inc. using standardprocedure.

PCR Amplification and Library Screening

The primers PLVAP Sac I F:

(SEQ ID NO: 36) 5′-CTCCAAGGTGGAGGAGCTGGC-3′and PLVAP Stop Bam HI R:

(SEQ ID NO: 37) 5′-GGATCCTGAGCATATCCCTGCATCCTCC-3′were used to amplify the sequence coding for the C-terminal portion ofthe PLVAP (amino acids 282-442) extracellular domain. PCR was performedusing the following thermal cycle: 94° C. for 5 min, followed by 35cycles of 94° C. for 30 s, 56° C. for 30 s and 72° C. for 60 s. DNA waspurified with the Qiaquick® PCR purification kit (Qiagen, Surrey, UK)and then used in the construction of a PLVAP Novatope® library accordingto the manufacturer's instructions (Novagen, Merck Biosciences Ltd.,Beeston, UK). Briefly, purified PCR product was digested with DNAse I inthe presence of Mn²⁺ and fragments between 50-150 base pairs (bp) weregel purified using a gel extraction kit (Qiagen). The DNA fragments wereend-filled using T4 DNA polymerase and Tth polymerase to add a single dAresidue to each 3′ end and then ligated into the pScreen 1b(+)T-vector(Novagen), which is designed to express small inserts as acarboxy-terminal fusion to the T7 bacterial phage gene 10 capsidprotein. The library was transformed into NovaBlue (DE3) cells andplated onto Luria-Bertani (LB) media plates containing carbenicillin (50μg/ml) and tetracycline (12.5 μg/ml). The resulting human PLVAP cDNAlibrary was screened (approximately 10⁴ colonies for each antibody)using the method described in the Novatope® manual: Colonies expressingPLVAP fragments were transferred to nitrocellulose filters by contactwith the colony for 1 min. Bacterial colonies on the filters were lysedby incubating in a sealed chloroform vapor chamber for 15 min. Proteinswere denatured in colony denaturing solution (20 mM Tris pH 7.9, 6 MUrea, 0.5 M NaCl) for 15 min and the filters were blocked for 30 min in1% (w/v) gelatin in TBS with 0.05% (v/v) Tween® 20 detergent (TBS-T).Colony debris was removed by wiping with a tissue and then the filterswere probed for 1 h with monoclonal antibody KFCC-GY4 and KFCC-GY5 (1μg/ml) in TBS-T with 0.5% (w/v) gelatin. Blots were washed for 15 mintwice in TBS-T with 0.5% (w/v) gelatin and then probed with a 1/5000dilution of goat-anti mouse immunoglobulin-alkaline phosphataseconjugate (Chemicon) in TBS-T with 0.5% (w/v) gelatin. After washing,blots were developed using bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium (BCIP: 33 μg/ml; NBT: 66 μg/ml) substrate (Sigma). Positivecolonies from each screen were plated onto fresh LB amp/tet plates andthen re-probed with monoclonal antibody. Confirmed positives were usedin DNA minipreps and the PLVAP gene inserts were sequenced using T7terminator primer.ELISA for Study of Binding Interference

Each well of an ELISA plate was coated with 50 μl KFCC-GY5 anti-PLVAPmonoclonal antibodies at a concentration of 5 μg/m in PBS overnight.After blocking and washing, 50 μl of recombinant PLVAP protein (0.5μg/ml) was added to each well. After incubation at room temperature for60 minutes, the wells were washed and 50 μl of biotinylated KFCC-GY4antibody at different concentrations was added to each well induplicates and incubated for 30 minutes. After three washings, the wellswere developed with 50 μl of diluted streptavidin-horseradish peroxidaseconjugate and substrate. Optical density at 450 nm was measured for eachwell. Each value was a mean of duplicates.

ELISA for Additive Binding

Each well of an ELISA plate was coated with 50 μl of 0.25 μg/mlrecombinant PLVAP protein. The wells were blocked and incubated with 50μl of each humanized antibody derived from KFCC-GY4 or KFCC-GY5, eitherseparately or together. The final concentration of each antibody was0.01 μg/ml. The values were an average of duplicates.

Results:

To characterize the KFCC-GY4 and KFCC-GY5 mAbs further, epitope mappingwas conducted to determine the antigenic sites in PLVAP that are boundby each of these antibodies. Initial results demonstrated that theantigenic epitopes for both antibodies reside in the C-terminal regionof PLVAP protein between amino acids 282 and 482 (FIGS. 28A, 28B and28C). Both KFCC-GY4 and KFCC-GY5 mAbs reacted positively withPLVAP₅₁₋₄₄₂ (lane 1) and PLVAP₂₈₂₋₄₄₂ (lane 2), but did not react withPLVAP51-292 (lane 3) or human CEACAM6 protein unrelated to PLVAP (lane4). The results indicate that the epitopes for both antibodies reside inthe C-terminal end of PLVAP between amino acid residues 292 to 442.

A finer mapping study revealed that the KFCC-GY4 mAb reacted with an E.coli clone expressing a peptide encompassing amino acids 431 to 442 ofhuman PLVAP and the KFCC-GY5 mAb reacted with an E. coli cloneexpressing a peptide encompassing amino acids 378 to 404 of human PLVAP.As depicted in Table 5, the epitopes for these two mAbs do not overlap.

TABLE 5 Antigenic epitopes for KFCC-GY4 and KFCC-GY5 monoclonal antibodies Amino acid sequence responsible for antigenic epitope for KFCC-GY5 mAb:Human 431 SQRPPAGIPVAPSSG             442 (SEQ ID NO: 38)Mouse 426 SQRLPVVNPAAQPSG             437 (SEQ ID NO: 39)Amino acid sequence responsible  for antigenic epitope for KFCC-GY4 mAb:Human 378 ELAIRNSALDTCIKTKSQPMMPVSRPM 404  (SEQ ID NO: 40)Mouse 375 EVDVRISALDTCVKAKSLPAVP-PRVS 400  (SEQ ID NO: 41)

The KFCC-GY4 and KFCC-GY5 mAbs could each bind to human PLVAP in anenzyme-linked immunoassay (ELISA) without interfering with the bindingof the other antibody (FIG. 29). This lack of interference was alsoobserved using fully humanized composite KFCC-GY4 and KFCC-GY5monoclonal antibodies (FIG. 30), which are described in more detailbelow.

Example 7 Production and Characterization of Chimeric Antibodies thatSpecifically Bind PLVAP

Materials and Methods:

Abbreviations

Abbreviation Description CDR Complementarity Determining region of anantibody variable region (numbered CDR1-3 for each of the heavy andlight chains, as defined by Kabat) Ec (0.1%) The Absorbance of a 1 mg/mlsolution of protein ELISA Enzyme linked immunosorbent assay FW Frameworkregion-scaffold region of a variable domain supporting the CDRs HRPHorse-radish peroxidase IgG Immunoglobulin G mAb Monoclonal antibodyOD280 nm Optical density measured at 280 nm P protein PLVAP protein PBSPhosphate-buffered saline TMB 3,3′,5,5′-tetramethylbenzidine V-regionVariable region of an antibody chainInitial Determination of Variable Region Sequences

Total RNA was extracted using Trizol® reagent (Invitrogen, Carlsbad,Calif.) from 2×10⁷ cultured hybridoma cells from the cell lines KFCC-GY4and KFCC-GY5. 5′ RACE was carried out using the FirstChoice® RLM-RACEkit (Ambion, Inc., Austin, Tex.) following the manufacturer'sinstructions to determine the coding sequence of VH and VL domains fromKFCC-GY4 and KFCC-GY5. Briefly, 10 μg of extracted RNA was treated withcalf intestinal phosphatase (CIP) in a 20 μl total volume reactionmixture containing 2 μl of 10×CIP buffer and 2 μl of CIP for 1 h at 37°C. After extracted with phenol/chloroform, RNA was precipitated withethanol and resuspended in 11 μl of nuclease-free water. 5 μl ofCIP-treated RNA was treated with tobacco acid pyrophosphatase (TAP) in a10 μl reaction mixture containing 1 μl of 10×TAP buffer and 2 μl of TAPfor 1 h at 37° C. 2 μl of the CIP/TAP-treated RNA was then ligated to300 ng of RNA adaptor by T4 RNA ligase in a 10 μL reaction mixture for 1h at 37° C. 2 μl of the ligated RNA or control RNA was used as atemplate to synthesize cDNA with M-MLV reverse transcriptase for 1 h at42° C. using random decamers. The cDNAs corresponding to variable heavy(VH) and light (VL) chains were then amplified by PCR separately withTakara Ex Taq™ DNA polymerase (Takara Bio Inc., Osaka, Japan) usingforward 5′ RACE outer primer 5′-GCTGATGGCGATGAATGAACACTG-3′ (SEQ IDNO:42) and reverse primers complementary to the nucleotide sequencesencoding the kappa chain constant region (5′-TCAACGTGAGGGTGCTGCTCATGC-3′(SEQ ID NO:43)) or the heavy chain CH1 region(5′-TTTCTTGTCCACCTTGGTGCTGCTGG-3′ (SEQ ID NO:44)), respectively. The PCRreaction mixtures were incubated for 5 min at 94° C. followed by 35amplification cycles, comprising denaturation at 94° C. for 30 s,annealing at 57° C. for 30 s and extension at 72° C. for 1 min. Thereaction was extended for another 7 min at 72° C. to insure fullextension. PCR products were analyzed and purified from the 1.5% agarosegel using the Qiaquick® gel extraction kit (Qiagen, Mississauga,Ontario, Canada). The purified PCR fragments were cloned into thepGEM®-T-easy plasmid vector (Promega, Madison, Wis., USA). A minimum of5 independent clones for each chain were subjected to nucleotidesequencing analysis. The CDRs were identified according to Kabatdefinition (FIGS. 15A, 15B, 16A and 16B).

Independent Confirmation of Variable Region Sequences

mRNA was successfully extracted (Promega Catalogue No. Z5400) fromfrozen KFCC-GY4 and KFCC-GY5 cells. RT-PCR was performed usingdegenerate primer pools for murine signal sequences with a singleconstant region primer. Heavy chain variable region mRNA was amplifiedusing a set of six degenerate primer pools (HA to HF) and light chainvariable region mRNA was amplified using a set of seven degenerateprimer pools (kappaA to kappaG)—monoclonal isotyping analysis of theIgGs using Roche Isostrips (Roche Catalogue No. 493027001) revealed themboth to be IgG1/kappa isotypes. Amplification products were obtainedwith heavy chain and kappa light chain primer pools confirming the lightchain is from the kappa cluster. Each product was cloned and severalclones from each sequenced. For KFCC-GY4 and KFCC-GY5 antibodies, singlefunctional heavy and light chain variable region sequences wereidentified for each antibody.

Expression of Chimeric Antibodies

The KFCC-GY4 and KFCC-GY5 variable regions were transferred to anexpression vector system (Antitope) for IgG4 heavy chain and kappa lightchain. NS0 cells were transfected via electroporation and selected usingmethotrexate. A number of methotrexate resistant colonies wereidentified and cell lines positive for IgG expression were expanded.Genomic DNA from the lead cell lines was recovered and subjected to PCRand confirmatory sequencing. IgG chimeric KFCC-GY4 and KFCC-GY5 IgG4swere purified from cell culture supernatants on a Protein A-Sepharose®column (GE Healthcare Catalogue No. 110034-93) and quantified by OD280nm using an extinction coefficient, Ec(0.1%), based on the predictedamino acid sequence—Ec(0.1%) values of 1.484 and 1.334 were used forchimeric KFCC-GY4 and KFCC-GY5, respectively. Greater than 2 mg ofantibody was purified and analyzed by SDS-PAGE (FIG. 31). Bandscorresponding to the predicted sizes of the heavy and light chains wereobserved with no evidence of any contamination.

Binding of Chimeric KFCC-GY4 and KFCC-GY5 Antibodies to RecombinantPLVAP

The binding of chimeric KFCC-GY4 and KFCC-GY5 antibodies to recombinantPLVAP protein was assessed in a competition ELISA. Either a dilutionseries of chimeric or control antibody from 30 μg/ml to 0.014 μg/ml(final concentration) was premixed with a constant concentration ofbiotinylated murine KFCC-GY4 (0.3 μg/ml, final concentration) or adilution series of chimeric or control antibody from 10 μg/ml to 0.004μg/ml (final concentration) was premixed with a constant concentrationof biotinylated murine KFCC-GY5 (0.1 μg/ml, final concentration) beforeincubating for 1 hour at room temperature on a Nunc Immuno™ MaxiSorp® 96well flat bottom microtitre plate (Fisher Catalogue No. DIS-971-030J)precoated with 1 μg/ml recombinant PLVAP protein diluted in PBS. Thebinding of the biotinylated mAb was determined by detection withstreptavidin-HRP and TMB substrate.

Results:

Variable regions from the KFCC-GY4 and KFCC-GY5 mouse anti-PLVAPmonoclonal antibodies were successfully cloned and sequenced. Threecomplementarity determining regions (CDRs) were identified in eachantibody according to Kabat definition. The analysis of the sequencesobtained from hybridomas KFCC-GY4 and KFCC-GY5 from the confirmationstudies are summarized in Tables 6 and 7. These sequences matched thesequences obtained by the initial characterization (FIGS. 15A, 15B, 16Aand 16B), with the exception of a single silent mismatch in the KFCC-GY4light chain nucleotide sequence. The aberrant transcript (GENBANK®accession number M35669) normally associated with the hybridoma fusionpartner SP2/0 was also detected in both cell lines.

TABLE 6 Sequence analysis of KFCC-GY4 monoclonal antibody H Chain LChain CDR 1 Length  5 aa 16 aa CDR 2 Length 17 aa  7 aa CDR 3 Length  4aa  9 aa Closest Human IGHV1-f*01 (64%) IGKV2D-30*01 (82%) Germline^(b)Closest Human FW1^(b) IGHV1-46*03 (68%) IGKV2D-29*02 (87%) Closest HumanFW2^(b) IGHV7-4-1*03 (71%) IGKV2D-30*01 (80%) Closest Human FW3^(b)IGHV1-f*01 (70%) IGKV2D-40*01 (94%) Closest Human J^(b) IGHJ6*01 (91%)IGKJ4 (90%) Max no. mouse FR 13 (4) 4 (1) residues^(c)

TABLE 7 Sequence analysis of KFCC-GY5 monoclonal antibody H Chain LChain CDR 1 Length  5 aa 16 aa CDR 2 Length 17 aa  7 aa CDR 3 Length  4aa  9 aa Closest Human IGHV1-46*03 (68%) IGKV2D-29*02 (80%) Germline^(b)Closest Human FW1^(b) IGHV7-4-1*02 (76%) IGKV2D-30*01 (83%) ClosestHuman FW2^(b) IGHV7-4-1*02 (79%) IGKV2D-40*01 (93%) Closest HumanFW3^(b) IGHV1-69*10 (72%) IGKV2D-40*01 (91%) Closest Human J^(b)IGHJ6*01 (91%) IGKJ2 (90%) Max no. mouse FR 9 (3) 2 (2) residues^(c)^(a)CDR definitions and sequence numbering according to Kabat^(b)Germline ID(s) indicated followed by % homology ^(c)Indicatesmaximum number of mouse residues that need to be sourced from humansequence segments with number of those potentially critical for affinityindicated in brackets

Variable region genes were then combined with human IgG4 heavy chain andkappa light chain constant regions and expressed in NS0 cells to producechimeric anti-PLVAP antibodies. To accomplish this, plasmid vectorscarrying KFCC-GY4 chimeric heavy and light chains and KFCC-GY5 chimericheavy and light chains were constructed (FIGS. 32A-1 to 32A-4, 32B-1 to32B-3, 32C-1 to 32C-3, 33A-1 to 33A-4, 33B-1 to 33B-3, 34A-1 to 34A-4,34B-1 to 34B-3, 34C-1 to 34C-4, 35A-1 to 35A-4 and 35B-1 to 35B-4).These plasmids were used to transfect NSO cells. Stably transfected andchimeric antibody-producing cells were cloned.

The heavy and light chain variable regions for the KFCC-GY4 antibodyshow good homology to their closest human germline sequences (64% and80%, respectively, for KFCC-GY4) and the individual framework sequenceshave close homologues in the human germline database. This thereforereduces the extent of engineering that needs to be undertaken for asuccessful humanized antibody. The maximum number of mouse frameworkresidues that will need to be sourced from human sequence segments forthe KFCC-GY4 heavy chain is 13, with 4 constraining residues probablybeing crucial for maintenance of binding activity. The maximum number ofmouse framework residues that will need to be sourced from humansequence segments for the KFCC-GY4 light chain is 4, with 1 constrainingresidue thought to be critical for activity (Table 6).

The heavy and light chain variable regions for the KFCC-GY5 antibodyalso showed good homology to their closest human germline sequences (68%and 80%, respectively, for KFCC-GY5) and the individual frameworksequences have close homologues in the human germline database. Thistherefore reduces the extent of engineering that needs to be undertakenfor a successful humanized antibody. The maximum number of mouseframework residues that will need to be sourced from human sequencesegments for the heavy chain is 9, with 3 constraining residues probablybeing crucial for maintenance of binding activity. The maximum number ofmouse framework residues that will need to be sourced from humansequence segments for the light chain is 2, with both thought to becritical for activity (Table 7). Composite Human Antibody™ analysis(Antitope; Cambridge, UK) revealed that human framework segments can befound to include all desirable mouse residues, and therefore completehumanized antibodies can be built from both templates.

A competition ELISA assay was used to demonstrate that the bindingefficiencies of the chimeric antibodies for PLVAP are similar to that ofthe respective parent murine antibodies. The chimeric IgG4-GY4 andmurine KFCC-GY4 antibodies had very similar binding profiles, with IC50values of 0.80 μg/m and 0.98 μg/ml, respectively (FIG. 36). Similarly,the chimeric IgG4-GY5 and murine KFCC-GY5 antibodies also had verysimilar binding profiles, with IC50 values of 0.40 μg/m and 0.49 μg/ml,respectively (FIG. 37). Therefore, the correct variable region sequencesfor the parent murine monoclonal antibodies were identified and cloned.

Binding affinities were also determined using Biacore™ system Bia T-100.Both chimeric KFCC-GY4 and KFCC-GY5 antibodies were determined to havehigher binding affinities for PLVAP than their respective parent mAbs(Table 8). The two chimeric monoclonal antibodies derived from KFCC-GY4and KFCC-GY5 are also referred to herein as CSR01 and CSR02,respectively.

TABLE 8 Binding affinities of KFCC-GY4, KFCC-GY5, chimeric KFCC-GY4(CSR01) and chimeric KFCC-GY5 (CSR02) monoclonal antibodies Antibody ka(1/Ms) kd (1/s) Kd (M) GY4 1.51 × 10⁴ 7.01 × 10⁻³ 4.64 × 10⁻⁷  ChimericGY4 5.21 × 10³ 2.12 × 10⁻³ 4.06 × 10⁻⁷  (CSR01) GY5 6.93 × 10³ 4.14 ×10⁻⁴ 5.98 × 10⁻⁸  Chimeric GY5 3.07 × 10⁴ 3.01 × 10⁻⁵ 9.78 × 10⁻¹⁰(CSR02) ka: association rate; kd: dissociation rate; Kd: dissociationconstant (binding affinity)

Example 8 Production and Characterization of Fully Humanized Antibodiesthat Specifically Bind PLVAP

Materials and Methods:

Overview

Three different kappa light chains and two different heavy chains wereconstructed based on the cDNA sequences from chimeric KFCC-GY4 (CSR01)antibody. They were used to transfect NSO cell line for production ofhumanized composite antibodies. Five different cell lines were generatedfor production of five monoclonal antibodies. Similarly, two differentkappa light chains and two different heavy chains were constructed basedon cDNA sequences from the chimeric KFCC-GY5 (CSR02) antibody. Four celllines that produce four different monoclonal antibodies were obtained.The nucleotide sequences of the variable domains of these humanizedheavy and light chains are summarized in FIGS. 38A-38E and 39A-39D. Aflowchart for derivation of chimeric antibodies and fully humanizedcomposite antibodies are outlined in FIG. 40.

Abbreviations

Abbreviation Description BLAST CDR Basic Local Alignment Search ToolComplementarity Determining region of an antibody variable region(numbered CDR1-3 for each of the heavy and light chains, as defined byKabat) Ec (0.1%) The absorbance of a 1 mg/ml solution of protein ELISAFW Enzyme linked immunosorbent assay Framework region-scaffold HRP IgGregion of a variable domain supporting the CDRs Horse-radish PeroxidaseImmunoglobulin G mAb Monoclonal antibody MHC Major histocompatibilitycomplex OD280 nm Optical density measured at 280 nm PBSPhosphate-buffered saline TMB 3,3′,5,5′-tetramethylbenzidine V-regionVariable region of an antibody chain P protein PLVAP proteinDesign of Human Antibody Variable Region Sequences

Structural models of the mouse KFCC-GY4 and KFCC-GY5 variable (V)regions were produced using Swiss PDB and analysed in order to identifyimportant “constraining” amino acids in the mouse V regions that werelikely essential for the binding properties of the antibody. Residuescontained within the CDRs (using both Kabat and Chothia definitions),together with a number of framework residues, were considered to beimportant. Both the VH and VK sequences of KFCC-GY4 and KFCC-GY5 containtypical framework residues. Whereas the CDR 1 and 2 motifs of bothantibodies are comparable to many murine antibodies, it was noted thatthe VH CDR3 for both antibodies are unusually short. From the aboveanalysis, it was considered that composite human sequences of bothKFCC-GY4 and KFCC-GY5 could be created with a wide latitude ofalternatives outside of CDRs, but with only a narrow menu of possiblealternative residues within the CDR sequences. Preliminary analysisindicated that corresponding sequence segments from several humanantibodies could be combined to create CDRs similar or identical tothose in the mouse sequences. For regions outside of and flanking theCDRs, a wide selection of human sequence segments were identified aspossible components of the novel human antibody variable regions.

Epitope Avoidance and Design of Variants

Based upon the above analysis, a large preliminary set of sequencesegments that could be used to create both KFCC-GY4 and KFCC-GY5 humanantibody variants was selected and analysed using iTope™ technology foranalysis of peptide binding to human MEW class II alleles, and using theTCED™ Cell Epitope Database of known antibody sequence-related T cellepitopes (Antitope Ltd.; Cambridge, UK). Sequence segments wheresignificant non-human MEW class II binding peptides were identified, orscored significant hits against the TCED™ database, were discarded. Thisresulted in a reduced set of segments, and combinations of these wereagain analysed, as above, to ensure that the junctions between segmentsdid not contain potential T cell epitopes. Selected segments were thencombined to produce heavy and light chain variable region sequences forsynthesis. For each antibody, five heavy chains and three light chainswere constructed with sequences as detailed in FIGS. 41A-41E and 42A-42C(for KFCC-GY5) and FIGS. 43A-43E and 44A-44C (for KFCC-GY4) and sequencealignments as detailed in FIGS. 45 and 46.

Construction, Expression and Purification of Variant Antibodies

Initial variant 1 human antibody VH and VK region genes were synthesizedfor KFCC-GY4 and KFCC-GY5 using a series of overlapping oligonucleotidesthat were annealed, ligated and PCR amplified to give full lengthsynthetic V regions. Subsequent human antibody sequence variants wereconstructed using long overlapping oligonucleotides and PCR, using theinitial variant 1 as the template. The assembled variants were thencloned directly into the pANT™ expression vector system (Antitope, Ltd.;Cambridge, UK) for IgG4 heavy chains and kappa light chains. Allcombinations of composite heavy and light chains (i.e., a total of 15pairings) were stably transfected into NS0 cells via electroporation andselected using 200 nM methotrexate (Sigma Catalogue No. M8407-500MG).Methotrexate resistant colonies for each construct were tested for IgGexpression levels and the best expressing lines were selected and frozenunder liquid nitrogen. IgG4 Variants for KFCC-GY4 and KFCC-GY5 werepurified from cell culture supernatants on a Protein A-Sepharose® column(GE Healthcare Catalogue No. 110034-93) and quantified by OD280 nm usingan extinction coefficient, Ec(0.1%), based on the predicted amino acidsequence (Table 9). Greater than 2 mg of antibody was purified andanalysed by SDS-PAGE (FIGS. 47A and 47B). Bands corresponding to thepredicted sizes of the heavy and light chains were observed with noevidence of any contamination.

TABLE 9 Ec (0.1%) values for KFCC-GY4 and KFCC-GY5 antibody variantsVariant Ec (0.1%) GY4 Chimera 1.48 GY4 Variants (all) 1.49 GY5 Chimera1.33 GY5 Variants VH4/VK2 1.33 VH5/VK2 GY5 Variants VH4/VK3 1.35 VH5/VK3

In addition, CHO-K1 cells were transiently transfected usingLipofectamine® 2000 (Invitrogen #11668-019). 72 hours aftertransfection, cell media was harvested for antibody purification.Briefly, IgG4 human antibody variants from transient transfections werepurified from cell culture supernatants on a Protein A-Sepharose® column(Sigma Catalogue No. P3391-1.5G) and quantified using an Fccapture/kappa chain detection ELISA (Sigma Catalogue No. 16260 andA7164) against a human IgG4/kappa standard (Sigma Catalogue No. 14639).A broad observation was that the number of expressing NS0 clones wassignificantly lower for KFCC-GY4 lineage clones when compared toKFCC-GY5 lineage clones. Furthermore, it was also noted that both stableand transient yields were lower for KFCC-GY4 lineage clones compared toKFCC-GY5. In some cases, the transient yields from KFCC-GY4 lineageswere not sufficient to allow complete characterization of the variants,and so all subsequent analysis was carried out using NS0 purifiedmaterial from stable cell lines.

Binding of the Variant Antibodies to Recombinant PLVAP Protein

The binding of KFCC-GY4 and KFCC-GY5 human antibody variants torecombinant PLVAP was assessed in a competition ELISA. Either a dilutionseries of variant or control antibody from 30 μg/ml to 0.014 μg/ml(final concentration) was premixed with a constant concentration ofbiotinylated murine KFCC-GY4 (0.3 μg/ml, final concentration) or adilution series of variant or control antibody from 10 μg/ml to 0.004μg/ml (final concentration) was premixed with a constant concentrationof biotinylated murine KFCC-GY5 (0.1 μg/ml, final concentration) beforeincubating for 1 hour at room temperature on a Nunc Immuno™ MaxiSorp® 96well flat bottom microtitre plate (Fisher Catalogue No. DIS-971-030J)precoated with 1 μg/ml recombinant “protein P” diluted in PBS. Thebinding of the biotinylated mAb was determined by detection withstreptavidin-HRP and TMB substrate. After stopping the reaction with 3MHCl, absorbance was measured at 450 nm on a Dynex Technologies MRX TC IIplate reader and the binding curves of the test antibodies comparedagainst the mouse reference standard.

Titration of PLVAP Binding by Chimeric and Fully Humanized CompositeAnti-PLVAP Monoclonal Antibodies

Each well of an ELISA plate was coated with 50 μl 2.5 μg/ml recombinantPLVAP protein in PBS. After washing and blocking, each well wasincubated with a different concentration of humanized monoclonalantibody (0.5 μg/ml to 0.0025 μg/ml) for 60 minutes at room temperature.The binding of the antibody to PLVAP was measured using alkalinephosphatase conjugate of mouse anti-human IgG4 monoclonal antibody (BDPharmingen) diluted 1000×.

Results:

The five lead variants of IgG4-GY4 and murine KFCC-GY4 antibodies havevery similar binding profiles (FIG. 48). Absolute IC50 values and valuesrelative to the KFCC-GY4 mAb for the five lead variants are shown inTable 10. All of the lead variants shown are within two-fold of theoriginal murine monoclonal antibody.

Similarly, the four lead variants of IgG4-GY5 and murine KFCC-GY5antibodies have very similar binding profiles (FIG. 49). Absolute IC50values and values relative to the KFCC-GY5 mAb for the five leadvariants are shown in Table 11. All of the lead variants shown arewithin two-fold of the original murine monoclonal antibody.

TABLE 10 IC50 Values of KFCC-GY4 Composite Human Antibody ™ VariantsIC₅₀ (ratio compared to GY4 Antibody IC₅₀ (μg/ml) mAb) GY4 mAB 1.82 1VH4/VK2 2.53 1.39 VH4/VK3 2.94 1.61 VH5/VK1 2.18 1.20 VH5/VK2 1.91 1.05VH5/VK3 2.82 1.55

GY4 Composite Human Antibody™ variants were purified from NS0 stablytransfected supernatant and tested in a competition assay withbiotinylated KFCC-GY4 mAb. IC₅₀ values are displayed and are alsonormalized against the binding of the reference KFCC-GY4 mAb.

TABLE 11 IC50 Values of KFCC-GY5 Composite Human AntibodyTM VariantsIC50 (ratio compared to GY5 Antibody IC50 (μg/ml) mAb) GY5 mAB 0.38 1VH4/VK2 0.56 1.47 VH4/VK3 0.43 1.13 VH5/VK2 0.60 1.58 VH5/VK3 0.41 1.08

GY5 Composite Human Antibody™ Variants were purified from NS0 stablytransfected supernatant and tested in a competition assay withbiotinylated KFCC-GY5 mAb. IC₅₀ values are displayed and are alsonormalized against the binding of the reference KFCC-GY5 mAb.

Composite humanized antibodies derived from chimeric KFCC-GY4 andKFCC-GY5 antibodies were able to bind to PLVAP protein (FIG. 50). Fullyhumanized composite antibodies from chimeric KFCC-GY4 (CSR01) bind lesswell than chimeric CSRO1. In contrast, fully humanized compositeantibodies from chimeric KFCC-GY5 (CSR02) bind more or less equally wellas chimeric CSR02.

To confirm that the fully humanized antibodies from KFCC-GY4 (CSR01) andKFCC-GY5 (CSR02) can bind to PLVAP without interfering with the bindingof each other, an in vitro enzyme linked immunoassay (ELISA) wasperformed to show that some of the fully humanized composite antibodiesfrom CSR01 and CSR02 are indeed additive in binding to PLVAP (FIG. 30).

Binding of the humanized antibodies to PLVAP proteins expressed on humanumbilical cord vascular endothelial cells was assessed byimmunofluorescence studies. All humanized antibodies tested in thesestudies were able to bind to endothelial cells (FIGS. 51A-51C, 52A-52Gand 53A-53E). These results indicate that the humanized antibodies thatbind these two epitopes can be used as therapeutic or diagnostic agentsindependently, or used together for their additive effect, if needed.

In summary, the results of the study described in the Examples hereindemonstrate that murine monoclonal antibodies can be developed againsttwo different well-defined antigenic epitopes in the extracellulardomain of the human PLVAP protein. The amino acid sequences of these twoepitopes have been identified. The binding of these antibodies to thetwo identified epitopes in PLVAP does not cause them to interfere witheach other and can be additive. Thus, these antibodies, and antibodiesderived from them, can be used as therapeutic or diagnostic agentsindividually, or used together for their additive effect, if needed. TheCDRs responsible for antigen binding have been identified. Thisinformation has been successfully utilized to humanize both anti-PLVAPmurine monoclonal antibodies for treatment of liver cancer in humansubjects and to reduce antigenicity.

Example 9 Development of a Diagnostic Assay for Detecting PLVAP Levelsin Serum

Materials and Methods:

PLVAP ELISA

-   -   1. Each well of an ELISA plate was coated with 50 μl KFCC-GY4        mAb at 5 μg/m overnight. The antibody was prepared in 1×PBS        buffer containing 0.02% sodium azide.    -   2. After washing each well with 200 ul washing buffer (PBS        containing 0.05% Tween-20) three times, each well was blocked        with 200 μl blocking buffer (washing buffer containing 2% bovine        serum albumin) for 30 minutes at room temperature.    -   3. Wells were washed three times after blocking.    -   4. 50 μl PLVAP standards and diluted serum samples were added        into designated wells in duplicates and incubated for 60 minutes        at room temperature. Standards and serum samples were diluted in        blocking buffer.    -   5. Wells were washed three times and 50 μl of biotinylated        KFCC-GY5 mAb were added at 0.25 μg/ml.    -   6. After incubation for 30 minutes, all wells were washed three        times.    -   7. 50 μl of 2500× diluted Streptavidin-horseradish peroxidase        (Pierce, Inc. catalog #: N100) were added to each well and        incubated for 30 minutes at room temperature.    -   8. After three washes, 100 μl OPD substrate prepared according        to manufacturer's instruction (Sigma, Inc. catalog #: P-6787)        were added and incubated for an optimal duration of time.    -   9. The incubation was stopped by adding 50 μl of 0.18M H₂SO₄.    -   10. OD measurements were taken at 570 nm.        Results:

Both murine KFCC-GY4 and KFCC-GY5 anti-human PLVAP monoclonal antibodieswere used to establish an enzyme-linked immunoassay (ELISA) to measurePLVAP protein concentration in serum. KFCC-GY4 antibody was used to coatan ELISA plate to capture PLVAP protein in serum, and biotinylatedKFCC-GY5 antibody was used to detect PLVAP protein captured by theKFCC-GY4 antibody. The recombinant PLVAP protein was used as a referencestandard. As shown by the standard curve of the PLVAP ELISA (FIG. 54),the sensitivity for this assay is about 50 ng/ml. When two serum samplesfrom two liver cancer patients were assayed in two dilutions (2× and4×), both had measurable PLVAP levels (450 ng/ml and 360 ng/nl) and wereparallel with the standard curve. No measurable PLVAP was detected inthe plasma of two healthy individuals. Therefore, this assay can be usedin diagnostic applications to assay PLVAP levels in serum.

The relevant teachings of all patents, published applications andreferences cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. An isolated nucleic acid encoding: a) at leastone heavy chain amino acid sequence selected from the group consistingof SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, and SEQ ID NO:102; b) at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:108; c) at least one heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86; or d) at least onekappa light chain amino acid sequence selected from the group consistingof SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90, and SEQ IDNO:92.
 2. The isolated nucleic acid of claim 1, wherein the isolatednucleic acid encodes: a) at least one heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, and SEQ ID NO:102; andb) at least one kappa light chain amino acid sequence selected from thegroup consisting of SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:104, SEQ ID NO:106, and SEQ ID NO:108.
 3. The isolated nucleic acidof claim 2, wherein the nucleic acid encodes a variable heavy domaincomprising an amino acid sequence comprising SEQ ID NO: 68 or 102; and avariable light domain comprising an amino acid sequence comprising SEQID NO: 62 or
 106. 4. The isolated nucleic acid of claim 1, wherein theisolated nucleic acid encodes: a) at least one heavy chain amino acidsequence selected from the group consisting of SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQID NO:86; or b) at least one kappa light chain amino acid sequenceselected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:88, SEQ ID NO:90, and SEQ ID NO:92.
 5. The isolated nucleic acid ofclaim 4, wherein the nucleic acid encodes a variable heavy domaincomprising an amino acid sequence comprising SEQ ID NO: 76 or 86; and avariable light domain comprising an amino acid sequence comprising SEQID NO: 72 or
 92. 6. An isolated cell comprising the nucleic acid ofclaim
 1. 7. An isolated cell comprising the nucleic acid of claim
 2. 8.An isolated cell comprising the nucleic acid of claim
 3. 9. An isolatedcell comprising the nucleic acid of claim
 4. 10. An isolated cellcomprising the nucleic acid of claim
 5. 11. An isolated cell comprising:i) a) a first nucleic acid encoding at least one heavy chain amino acidsequence selected from the group consisting of SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, and SEQID NO:102; and b) a second nucleic acid encoding at least one kappalight chain amino acid sequence selected from the group consisting ofSEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106,and SEQ ID NO:108; or ii) a) a first nucleic acid encoding at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, and SEQ ID NO:86; and b) a second nucleic acid encoding atleast one kappa light chain amino acid sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90,and SEQ ID NO:92.
 12. An isolated protein comprising: i) a) at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, and SEQ ID NO:102; and b) at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:108; or ii) a) at least one heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86; and b) atleast one kappa light chain amino acid sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90,and SEQ ID NO:92.
 13. The protein of claim 12, wherein the protein isbound to a radioactive isotope or a cytotoxic agent.
 14. The protein ofclaim 12, wherein the protein is a monoclonal antibody that specificallybinds SEQ ID NO:
 23. 15. A composition comprising the protein of claim12.
 16. The composition of claim 15, further comprising at least onechemotherapeutic agent.
 17. The composition of claim 16, wherein thechemotherapeutic agent is selected from the group consisting ofdoxorubicin, cisplatin, mitomycin, 5-fluorouracil, tamoxifen, sorafeniband octreotide.
 18. A method of making a protein, comprising culturingthe cell of claim 6 under conditions to express the protein andisolating the protein, wherein the protein comprises: i) a) at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, and SEQ ID NO:102; and b) at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:108; or ii) a) at least one heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86; and b) atleast one kappa light chain amino acid sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90,and SEQ ID NO:92.
 19. A method of making a protein, comprising culturingthe cell of claim 7 under conditions to express the protein andisolating the protein, wherein the protein comprises: i) a) at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, and SEQ ID NO:102; and b) at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:108; or ii) a) at least one heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86; and b) atleast one kappa light chain amino acid sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90,and SEQ ID NO:92.
 20. A method of making a protein, comprising culturingthe cell of claim 11 under conditions to express the protein andisolating the protein, wherein the protein comprises: i) a) at least oneheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, and SEQ ID NO:102; and b) at least one kappa light chainamino acid sequence selected from the group consisting of SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:108; or ii) a) at least one heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86; and b) atleast one kappa light chain amino acid sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:88, SEQ ID NO:90,and SEQ ID NO:92.